Compositions and Methods for Treating Pathological Calcification and Ossification

ABSTRACT

The present invention includes compositions and methods for treating disease and disorders associated with pathological calcification or pathological ossification by modulating the level or activity of NPP1 or a mutant thereof, or a mutant NPP4 modified to exhibit ATP hydrolase activity similar to the hydrolase activity of NPP1.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 16/033,948, filed Jul. 12, 2018, nowallowed, which is a continuation of, and claims priority to, U.S. patentapplication Ser. No. 15/686,972, filed Aug. 25, 2017, now issued as U.S.Pat. No. 10,052,367, which is a continuation of, and claims priority to,U.S. patent application Ser. No. 14/765,514, filed Aug. 3, 2015, nowissued as U.S. Pat. No. 9,744,219, which is a 35 U.S.C. § 371 nationalphase application from, and claiming priority to, InternationalApplication No. PCT/US2014/015945, filed Feb. 12, 2014, and publishedunder PCT Article 21(2) in English, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/904,786, filedNov. 15, 2013, and No. 61/764,297, filed Feb. 13, 2013, all of whichapplications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Ectopic tissue mineralization is associated with numerous humandiseases, including chronic joint disease and acutely fatal neonatalsyndromes. To prevent unwanted tissue calcification, factors whichpromote and inhibit tissue mineralization must be kept in tight balance.Genetic analysis of human kindreds and animal models of diseasesassociated with ectopic calcification have identified the balance ofextracellular inorganic pyrophosphate (PPi) and phosphate (Pi) as animportant regulator of ectopic tissue mineralization (Terkeltaub, 2001,Am. J. Phys. Cell Phys., 281:C1-C11). The activity of threeextracellular enzymes—Tissue Non-specific Alkaline Phosphatase (TNAP),Progressive Ankylosis Protein (ANK), and Ecto-NucleotidePyrophosphatase/Phosphodiesterase-1 (NPP1)—tightly control theconcentration of Pi and PPi in mammals at 1-3 mM and 2-3 μMrespectively. PPi is a regulator of biomineralization, inhibiting theformation of basic calcium phosphate from amorphous calcium phosphate.

Diseases of ectopic calcification range from ultra-rare fatal diseasesof infancy to common ailments associated with aging in a largepercentage of the human population. IIAC, also referred to asgeneralized arterial calcification of infancy (GACI), is a rare andfatal form of ectopic calcification present in a very small cohort ofunrelated kindreds (approximately 200 reported cases), and ischaracterized by the calcification of the internal elastic lamina ofmuscular arteries and stenosis due to myointimal proliferation. Whilethe clinical presentation of these patients is variable, this maladyresults in death in the neonatal period, usually by age 6 months. OPLLis a common form of human mylelopathy caused by a compression of thespinal cord by ectopic ossification of the spinal ligaments (Stapletonet al., 2011. Neurosurgical Focus 30:E6). The disease occurs mostfrequently in the cervical spine, and was first described in theJapanese population where it has a prevalence of 1.9-4.3% of the entirepopulation. The disease presentation is also variable, but several genesand proteins have emerged over the years as promising targets ofetiologic investigation.

The human NPP family consists of seven extracellular, glycosylatedproteins (i.e., NPP1, NPP2, NPP3, NPP4, NPP5, NPP6, and NPP7) thathydrolyze phosphodiester bonds (Bollen et al., 2000, Crit. Rev. Biochem.Mol. Biol. 35:393-432; Stefan et al., 2005, Trends Biochem. Sci.30:542-550; Goding et al., 2003, Biochim. Biophys. Acta 1638:1-19). Theenzymes are numbered in the order they were discovered. NPPs arecell-surface enzymes, with the exception of NPP2, which is exported tothe plasma membrane but cleaved by furin and released into theextracellular fluid (Jansen et al., 2005, J. Cell Sci. 118:3081-3089).The enzymes have high degrees of sequence and structural homology, butexhibit a diverse substrate specificity that encompasses nucleotides tolipids.

NPP1 (also known as PC-1) is a type 2 extracellular membrane-boundglycoprotein located on the mineral-depositing matrix vesicles ofosteoblasts and chondrocytes, and hydrolyzes extracellular nucleotides(principally ATP) into AMP and PPi (Bollen et al., 2000, Crit. Rev.Biochem. Mol. Biol. 35:393-432; Terkeltaub, 2006, Purinergic signaling2:371-377). PPi functions as a potent inhibitor of ectopic tissuemineralization by binding to nascent hydroxyapatite (HA) crystals,thereby preventing the future growth of these crystals (Terkeltaub,2006, Purinergic signaling 2:371-377; Addison et al., 2007, J. Biol.Chem. 282:15872-15873). NPP1 generates PPi via the hydrolysis ofnucleotide triphosphates (NTP's), ANK transports intracellular PPi intothe extracellular space, and TNAP removes PPi via the direct hydrolysisof PPi into Pi (FIG. 1).

NPP2 is a lysophospholipase-D enzyme that generates lyso-phosphatidicacid (LPA) from lyso-phosphocholine (Umezu-Goto et al., 2002, J. CellBiol. 158:227-233). NPP4 was recently shown to be a di-adenosinetriphosphate (Ap3A) hydrolase and a potent pro-coagulant factor on thesurface of vascular surfaces (Albright et al., 2012, Blood120:4432-4440). NPP5 remains uncharacterized, and NPP6 and NPP7 bothhydrolyze lipid substrates; NPP6 is a lysophopholipase-C enzyme, andNPP7 is an alkaline sphingomyelinase (Duan et al., 2003, J. Biol. Chem.278:38528-36; Sakagami et al., 2005, J. Biol. Chem. 23084-93).

The lack of production and purification of significant quantities ofbiologically active NPP proteins in this membrane bound protein familyhas previously hampered their study and characterization. Expressionsystems for soluble NPP4 and NPP1 have not been demonstrated for largescale protein production and purification. Mutations in NPP4 to alterthe enzymatic activity of the enzyme from Ap3A to ATP have not beenreported.

Extracellular nucleotides engage in paracrine and autocrine cellsignaling by binding purinergic receptors on cell surfaces, resulting ina wide range of physiologic responses including platelet aggregation(Offermanns, 2006, Circ. Res. 99:1293-1304), bone development andremodeling (Terkeltaub, 2006, Purinergic Signalling 2:371-377), andendocrinopathies such as diabetes and obesity (Omatsu-Kanbe et al.,2002, Exper. Physiol. 87:643-652; Schodel et al., 2004, Biochem.Biophys. Res. Comm. 321:767-773). Purinergic P2X receptors present oncell surfaces are ion channels that bind mainly ATP, while P2Y receptorsare cell surface G-protein coupled receptors that interact with abroader range of nucleotides. The concentration of extracellular purinesubstrates driving purinergic signaling is determined by the release ofectonucleotides via degranulation or cell lysis, the rate ofectonucleotide synthesis, and the catabolism of ectonucleotides byectoenzymes.

Platelet aggregation is induced via interaction of extracellular ADPwith platelet P2Y₁ and P2Y₁₂ purinergic receptors, resulting in rapidcalcium influx followed by further platelet activation, degranulation,and irreversible shape change to extend the growing thrombus. Metabolismof extracellular ADP by membrane-bound CD39 on vascular endothelialcells and soluble phosphohydrolases in the platelet microenvironmentrapidly degrade ADP into AMP and Pi, limiting the extension of theaggregatory burst of ADP to platelets in the immediate vicinity of theactivated, degranulating platelets. AMP is further metabolized bymembrane bound CD73 into adenosine, a potent antithrombotic signalingmolecule which modulates vascular tone, decreases leukocyte adhesion,and limits thrombus formation. The release of platelet dense coregranules disgorges high concentrations of ADP into the thromboticmicroenvironment, further stimulating platelet aggregation.

Platelets dense-core granules also contain high concentrations of thedinucleotide Ap3A, which can reach local concentrations of over 100 μMupon platelet degranulation. The role of Ap3A in hemostasis has neverbeen fully defined, but Ap3A has long been thought to represent morestable ‘chemically masked’ ADP which could be released into thethrombotic microenvironment to sustain platelet aggregation. Ap3Ahydrolytic activity has been identified on the vascular surfaces of bothbovine and porcine endothelial cells.

There is a need in the art for novel compositions and methods fortreating diseases and disorders associated with pathologicalcalcification and/or pathological ossification. Such compositions andmethods should not undesirably disturb other physiologic processes. Thepresent invention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of treating or preventinga disease or disorder associated with pathological calcification orpathological ossification in a subject in need thereof, wherein themethod comprises administering to the subject a therapeuticallyeffective amount of a composition comprising at least one agent selectedfrom the group consisting of an ecto-nucleotidepyrophosphate/phosphodiesterase-1 (NPP1) polypeptide and a fragment,derivative, mutant or mutant fragment thereof, and an activator of NPP1polypeptide and a fragment, mutant or mutant fragment thereof, wherebythe disease or disorder is treated or prevented in the subject.

In another aspect, the invention includes a method of treating a diseaseor disorder associated with pathological calcification or pathologicalossification in a subject in need thereof, wherein the method comprisesadministering to the subject a therapeutically effective amount of acomposition comprising at least one agent selected from the groupconsisting of an ecto-nucleotide pyrophosphate/phosphodiesterase-4(NPP4) polypeptide and a fragment, derivative, mutant or mutant fragmentthereof, and an activator of NPP4 polypeptide or fragment or mutantthereof.

In yet another aspect, the invention includes a composition comprisingat least one agent selected from the group consisting of anecto-nucleotide pyrophosphate/phosphodiesterase-1 (NPP1) polypeptide, anNPP1 polypeptide fragment, an NPP1 polypeptide derivative, a mutant NPP1polypeptide, and a mutant NPP1 polypeptide fragment.

In yet another aspect, the invention includes a composition comprisingat least one agent selected from the group consisting of anecto-nucleotide pyrophosphate/phosphodiesterase-4 (NPP4) polypeptide, anNPP4 polypeptide fragment, an NPP4 polypeptide derivative, a mutant NPP4polypeptide, and a mutant NPP4 polypeptide fragment.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the NPP1 polypeptide or a fragment,mutant or mutant fragment thereof comprises a soluble recombinant NPP1polypeptide or a fragment, mutant or mutant fragment thereof. In certainembodiments of the aspects recited herein, the NPP1 polypeptide or afragment, mutant or mutant fragment thereof lacks the NPP1 transmembranedomain. In other embodiments of the aspects recited herein, the NPP1polypeptide or a fragment, mutant or mutant fragment thereof comprisesan IgG Fc domain. In yet other embodiments of the aspects recitedherein, the mutant NPP1 polypeptide or fragment thereof has lower Ap3Ahydrolytic activity as compared to the corresponding wild-type NPP1polypeptide or fragment thereof. In yet other embodiments of the aspectsrecited herein, the mutant NPP1 polypeptide or fragment thereof hassubstantially the same ATP hydrolytic activity as compared to thecorresponding wild-type NPP1 polypeptide or fragment thereof. In yetother embodiments of the aspects recited herein, the mutant NPP1polypeptide or fragment thereof has lower Ap3A hydrolytic activity andsubstantially the same ATP hydrolytic activity as compared to thecorresponding wild-type NPP1 polypeptide or fragment thereof. In yetother embodiments of the aspects recited herein, the mutant NPP1polypeptide or fragment thereof has a mutation in at least one positionselected from the group consisting of Ser 532, Tyr 529, Tyr 451, Ile450, Ser 381, Tyr 382, Ser 377, Phe 346, Gly 531, Ser 289, Ser 287, Ala454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp 276, Tyr434, Gln 519, Ser 525, Gly 342, Ser 343 and Gly 536, relative to SEQ IDNO:1.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the NPP1 polypeptide or a fragment,mutant or mutant fragment thereof comprises a polyaspartic acid domain.In certain embodiments of the aspects recited herein, the polyasparticacid domain comprises from about 2 to about 20 or more sequentialaspartic acid residues. In other embodiments of the aspects recitedherein, the NPP1 polypeptide or a fragment, mutant or mutant fragmentthereof comprises an NPP2 transmembrane domain. In yet other embodimentsof the aspects recited herein, the activator of NPP1 activates at leastone selected from the group consisting of: expression of NPP1polypeptide or a fragment, mutant or mutant fragment thereof; andactivity of NPP1 polypeptide or a fragment, mutant or mutant fragmentthereof. In yet other embodiments of the aspects recited herein, theactivator of NPP1 is at least one selected from the group consisting ofa chemical compound, a protein, a peptide, a peptidomimetic, and a smallmolecule chemical compound.

In certain embodiments of the aspects recited herein, the at least oneagent is administered acutely or chronically. In other embodiments ofthe aspects recited herein, the at least one agent is administeredlocally, regionally or systemically. In yet other embodiments of theaspects recited herein, the subject is human. In yet other embodimentsof the aspects recited herein, the disease or disorder is at least oneselected from the group consisting of idiopathic infantile arterialcalcification (IIAC), ossification of the posterior longitudinalligament (OPLL), hypophosphatemic rickets, osteoarthritis, andcalcification of atherosclerotic plaques.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the NPP4 polypeptide or a fragment,mutant or mutant fragment thereof is a soluble recombinant NPP4polypeptide or a fragment, mutant or mutant fragment thereof. In certainembodiments of the aspects recited herein, the mutant NPP4 polypeptideor fragment thereof comprises at least one mutation selected from thegroup consisting of D335, S92, D264, L265, S330, Q331, K332, and T323,relative to SEQ ID NO:3. In other embodiments of the aspects recitedherein, the mutant NPP4 polypeptide or fragment thereof comprises atleast one mutation which increases the ATP hydrolytic activity of themutant NPP4 polypeptide or fragment thereof as compared to thecorresponding wild-type NPP4 or fragment thereof. In yet otherembodiments of the aspects recited herein, the mutant NPP4 polypeptideor fragment thereof comprises at least one mutation which increases theNPP1-like hydrolytic activity of the mutant NPP4 polypeptide or fragmentthereof as compared to the corresponding wild-type NPP4 or fragmentthereof. In yet other embodiments of the aspects recited herein, themutant NPP4 polypeptide or fragment thereof comprises at least onemutation which increases the substrate selectivity of the mutant NPP4polypeptide for ATP as compared to the corresponding wild-type NPP4 orfragment thereof. In yet other embodiments of the aspects recitedherein, the mutant NPP4 polypeptide or fragment thereof comprises atleast one mutation which decreases the Ap3A hydrolytic activity of theenzyme as compared to the corresponding wild-type NPP4 or fragmentthereof. In yet other embodiments of the aspects recited herein, themutant NPP4 polypeptide or fragment thereof comprises at least onemutation which substantially increases the ATP hydrolysis activity anddecreases the Ap3A hydrolytic activity of the enzyme as compared to thecorresponding wild-type NPP4 or fragment thereof. In yet otherembodiments of the aspects recited herein, the NPP4 polypeptide or afragment, mutant or mutant fragment thereof lacks the NPP4 transmembranedomain. In yet other embodiments of the aspects recited herein, the NPP4polypeptide or a fragment, mutant or mutant fragment thereof comprisesan IgG Fc domain.

In various embodiments of any of the above aspects or any other aspectof the invention delineated herein, the NPP4 polypeptide or a fragment,mutant or mutant fragment thereof comprises a polyaspartic acid domain.In certain embodiments of the aspects recited herein, the polyasparticacid domain comprises from about 2 to about 20 or more sequentialaspartic acid residues. In other embodiments of the aspects recitedherein, the activator of NPP4 activates at least one selected from thegroup consisting of: expression of NPP4 polypeptide or a fragment,mutant or mutant fragment thereof; and activity of NPP4 polypeptide or afragment, mutant or mutant fragment thereof. In yet other embodiments ofthe aspects recited herein, the activator of NPP4 is at least oneselected from the group consisting of a chemical compound, a protein, apeptide, a peptidomimetic, and a small molecule chemical compound.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an illustration depicting proteins regulating theextracellular balance of inorganic pyrophosphate (PPi) and phosphate(Pi). Inorganic pyrophosphate (PPi) is generated by the cleavage ofextracellular nucleotide triphosphates (NTPs) by NPP1 or the transfer ofPPi from the intracellular to extracellular space by Ank. TNAP degradesPPi to generate Pi.

FIGS. 2A-2B are a set of illustrations depicting the cloning strategy ofNPP1. FIG. 2A: NPP1 is replaced with the signal peptide from NPP2 so itcan be expressed as a secreted protein. The NPP1 fragments on eitherside of the transmembrane region (indicated by 1 and 2) are PCRamplified separately and substituted with the NPP2 signal peptidesequence (indicated by 3, by the arrow). FIG. 2B: Schematic figure ofdomain architecture and expressed proteins used in this study. NPP1 is atype II transmembrane protein, while NPP4 is a type I transmembraneprotein. The proteins are represented by schematics colored toillustrate the domain architecture—transmembrane domains are indicatedas 1, somatomedin B domains are indicated as 2, catalytic domains areindicated as 3, the nuclease domain is indicated as 4, and the signalpeptide of NPP4 is indicated as 5. The expressed NPP1 protein consistsof residues 96-925 of the human sequence, comprising the entireextracellular sequence of the protein, containing both somatomedin Bdomains, the catalytic domain, and the nuclease domain of the protein.The secreted NPP4 protein also consists of the entire extracellularportion of the protein, comprising amino acids 16-407 of the humansequence.

FIG. 3 is a photograph of a gel depicting the expression andpurification of NPP1 in Baculovirus. Baculovirus cells were infectedwith NPP1 virus, and the extracellular media was collected following twodays of incubation, concentrated, and run over a nickle column.Following washing with buffer, the protein was eluted with imidazole and5 ml fractions were collected and run on an SDS-PAGE gel (E1-E9).

FIG. 4 is a photograph of a gel depicting the expression andpurification of NPP1 in mammalian cells: To express NPP1 with mammalianglycosylations, NPP1 was produced in HEK293 kidney cells using ananalogous expression and purification procedure which has been describedfor baculovirus. The SDS-PAGE gel of the purified protein is depicted.The concentration of the NPP1 stock solution as determined by amino acidanalysis is 2.15 mg/ml.

FIGS. 5A-5E illustrate the results of experiments assessing ATP cleavageand hydrolysis. FIG. 5A is a graph depicting the HPLC analysis of ATPcleavage by NPP1 (nM NPP1 supplemented with 500 μM ATP quenched at (frombottom to top) 3, 6, and 30 minutes). The enzymatic products of ATPhydrolysis by NPP1 were confirmed by HPLC. FIG. 5B is a graph depictingthe comparison of ATP hydrolysis between NPP1 and NPP4. The comparisonof the ATP hydrolytic activity of NPP1 and NPP4 (which share 38%sequence identity) reveals that only NPP1 hydrolyzes ATP, revealing thetuned substrate specificity of the NPP family. FIG. 5C is a graphdepicting the results of experiments assessing steady-state ATP cleavageby NPP1. Time courses of ATP cleavage monitored at absorbance 259 nmafter mixing 200 nM of NPP1 were used to derive the initial ratevelocities at each ATP concentration, and the data is fit to arectangular hyperbola. The smooth line through the data is the best fitto a hyperbola, resulting in K_(M)=144.5 (±36.0) μM and k_(cat)=468(±48) min⁻¹=7.8 (±0.8) s⁻¹. FIG. 5D: Ap3A concentration dependence ofthe NPP1 and NPP4 initial steady state Ap3A substrate cleavage rateobtained from the best linear fits of absorption change of time courses.The smooth lines though the data are the best fit to a hyperbola withK_(M)=20 (±3) μM and k_(cat)=7.2 (±0.3) s⁻¹ NPP⁻¹ for NPP1, while withK_(M)=685 (±108) μM and k_(cat)=8.0 (±0.1) s⁻¹ NPP⁻¹ for NPP4. FIG. 5E:Inhibition of NPP4 cleavage activity by nucleotide monophosphates. Thesolid line through the data points represent the best fit to arectangular hyperbola. Symbols: AMP: magenta diamond, CMP: black square,GMP: blue circle. The resulting IC₅₀, from strongest to weakestinhibition, is AMP 129±73 μM, CMP 322±39 μM, UMP 2.11±0.37 mM (not shownfor clarity), and GMP 2.98±0.38 mM.

FIG. 6A-6C are a series of illustrations relating to NPP4-AMP structureand comparison to other NPP catalytic domains. The three dimensionalstructure of NPP4 with enzymatic product (AMP) bound was determined to1.5 Å resolution by X-ray crystallography. FIG. 6A: The protein isdisplayed as a ribbon with AMP in stick form colored by atom type.Active site zinc ions are depicted as spheres. FIG. 6B: Superposition ofCα-traces of human NPP4 with other NPP catalytic domains reveals thatthe greatest degree of structural conservation (red, rmsd of 0.68 Å forall 4 molecules as shown) occurs throughout the central β-sheet core andthe active site near the zinc ions, including the α-helix on which thecatalytic threonine is located and the backbone near the hydrophobicslot. Regions of moderate similarity are yellow (rmsd of 1.33 Å) andthose of the lowest similarity are cyan (rmsd of 2.51 Å). The ccp4program Superpose was used to overlay the conserved subdomain(red-yellow, rmsd of 1.17 Å). Despite the high degree of structuralconservation near the active site, different NPPs can display widelyvarying substrate specificities. The four superposed catalytic domainsare from human NPP4 (presented here), mouse NPP1 (4B56), mouse NPP2(3NKN), and a bacterial NPP (2GSU). To accommodate a lipid substrate,NPP2 lacks a region found in all other NPP catalytic domains andtherefore an 8-residue linker (residues 272 to 279) within mouse NPP2was omitted in this comparison figure. Superposition of the entirecatalytic domain of NPP4 with that of NPP1, NPP2 and the bacterial NPPyield rmsd values of 1.54 Å, 1.43 Å and 1.43 Å, respectively. FIG. 6C:NPP4 product complex with AMP. Omit mFo-DFc difference-density is shownfor each, contoured at 3a. Ligands and water molecules within thebinding pocket were not included in the electron density mapcalculations. PyMOL was used to generate all molecular images (MolecularGraphics System, Version 1.2r3pre, Schrödinger, LLC).

FIGS. 7A-7D illustrate the base recognition of substrate at the NPP4catalytic site. FIG. 7A: The active site of NPP4 contains a pre-formedhydrophobic slot that results in its specificity for 5′ nucleotidecontaining substrates. In the NPP4-AMP complex, the adenine ring stackswith Tyr154 on one wall of the pocket while receiving favorable VDWinteractions from the tip of Phe71 along the opposite wall. To displaythe fit, the molecular surface of NPP4 (in mesh) and the VDW surface ofAMP (in semi-transparent shape) are shown. FIG. 7B: Position of AMP inthe active site with key residues and bound-metal ions highlighted. Thetwo bound zinc ions (spheres) play different roles, with Zn2 serving toactivate Thr70 (green) for nucleophilic attack on the substrate, whileZn1 electrostatically draws a phosphate group of the substrate intoclose proximity. Tyr154 and Phe71 of the nucleotide slot are cyan. FIG.7C: Superposition of the NPP4-AMP complex with apo NPP4 to illustratethat there is very little change when product is bound. A citrate anionbound at Zn1 of the apo structure has been omitted for visual clarity.FIG. 7D: Superposition of NPP4-AMP and NPP1-AMP complexes to illustratethe similar geometry within this half of their active sites, with bothpossessing a slot for nucleotide binding.

FIG. 8 is a scheme illustrating a non-limiting enzymatic mechanism ofAp3A hydrolysis by NPP4. Proposed reaction mechanism for NPP4 hydrolysisof Ap3A, based on active site homology to APs as originally proposed byGijsbers et al. Boxes have been placed around the steps in the mechanismfor which crystal structures have been obtained.

FIGS. 9A-9F illustrate a non-limiting modeling of the molecular basis ofsubstrate discrimination of NPP1 and NPP4. Models of ATP bound in anAMP-like orientation are shown for NPP1 (left column) and NPP4 (rightcolumn), based on the AMP cocrystal structures for each enzyme. FIG. 9A:In NPP1, the γ-phosphate of ATP is simultaneously stabilized by threelysine residues, two of which line the upper edge of the pocket andbecome ordered only when substrate is present due to electrostatics. Asa result of this tri-partite lysine claw, the γ-phosphate of bound ATPis favorably charge-stabilized and largely shielded from solvent by aninduced-fit lid comprised of the long hydrophobic side chains of thesetwo lysines along with an adjacent tyrosine ring. In contrast, NPP4offers a significantly less favorable γ-phosphate environment for asimilarly bound ATP, with less charge-stabilization, a more openarchitecture with no lid mechanism, and two nearby aspartate residuesfor charge-repulsion. As a result, ATP is not likely to bind in thisorientation to NPP4 very often. FIGS. 9A & 9B: Stick figures of ATPbound as modeled from AMP cocrystal structures. FIGS. 9C & 9D: Same, butas a molecular surface with tips of nearby charged side chains (positiveor negative, as indicated). FIGS. 9E & 9F: Rotated about 90 degrees.Sequence alignments show that human NPP1 retains all of the featuresderived from the mouse NPP1 structure.

FIGS. 10A-10B are a series of graph illustrating the effect of NPP1 inplatelet aggregation. Light transmission aggregometry was used to assessplatelet aggregation in response to increasing concentrations of NPP1and NPP4 and 80 μM Ap3A in platelet rich plasma. Data are showngraphically as percent of light transmittance (y-axis) over time(x-axis). FIG. 10A: In the absence of NPP1, 80 μM Ap3A elicited only aprimary wave of aggregation followed by rapid disaggregation, and thispattern was similarly observed with the addition of NPP1 inconcentrations of 300 pM and 500 pM. In contrast, 1 nM NPP1 in thepresence of 80 uM Ap3A stimulated a measurable secondary wave ofaggregation. FIG. 10B: NPP4 exhibited primary aggregation in thepresence of either Ap3A alone or Ap3A containing 1 nM NPP4. In thepresence of 20 nM NPP4 and higher there was marked secondary plateletaggregation. The findings suggest that either protein may directlystimulates platelet aggregation at low nM concentrations in the presenceof physiologic concentrations of Ap3A.

FIG. 11 is an illustrative model of Ap3A docketed into human NPP4.

FIG. 12 is a set of photographs illustrating protein crystals comprisingan inactive form of NPP4 bound to Ap3A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that an NPP1 polypeptide,fragment, derivative, mutant, or mutant fragment thereof, and a mutantNPP4 polypeptide or fragment thereof, are useful for the treatment ofdiseases and disorders involving pathological calcification and/orossification.

Thus, in certain embodiments, the invention relates to compositions andmethods for increasing the level or activity of an NPP1 polypeptide,fragment, derivative, mutant, or mutant fragment thereof, while in otherembodiments the invention relates to compositions and methods forincreasing the level or activity of a mutant NPP4 polypeptide, fragment,or derivative thereof. In yet other embodiments, the invention relatesto compositions and methods for increasing the level or activity of anNPP1 polypeptide, fragment, derivative, mutant, or mutant fragmentthereof, and a mutant NPP4 polypeptide or fragment thereof.

In certain embodiments, the invention relates to a method of eliminatingand/or reducing the pro-thrombotic activity, while retaining the ATPhydrolytic activity, of at least one agent selected from the groupconsisting of an ecto-nucleotide pyrophosphate/phosphodiesterase-1(NPP1) polypeptide and a fragment, derivative, mutant or mutant fragmentthereof. In other embodiments, the invention relates to a method ofeliminating and/or reducing the pro-thrombotic activity, while alsoincreasing the ATP hydrolytic activity, of at least one agent selectedfrom the group consisting of an ecto-nucleotidepyrophosphate/phosphodiesterase-1 (NPP4) polypeptide and a fragment,derivative, mutant or mutant fragment thereof. In yet other embodiments,the methods of the invention allow for safely treating ectopicmineralization without inducing unintended risks associated with apro-thrombotic state.

In various embodiments, the mutant NPP1 polypeptide, fragment orderivative thereof useful within the methods of the invention has lowerAp3A hydrolytic activity as compared to the corresponding wild-type NPP1polypeptide, fragment or derivative thereof. In various embodiments, themutant NPP1 polypeptide, fragment or derivative thereof useful withinthe methods of the invention has substantially the same ATP hydrolyticactivity as compared to the corresponding wild-type NPP1 polypeptide,fragment or derivative thereof. In various embodiments, the mutant NPP1polypeptide, fragment or derivative thereof useful within the methods ofthe invention has lower Ap3A hydrolytic activity and substantially thesame ATP hydrolytic activity as compared to the corresponding wild-typeNPP1 polypeptide, fragment or derivative thereof.

In various embodiments, the invention relates to compositions andmethods for increasing the level or activity of NPP1 polypeptide,fragment, derivative, mutant, or mutant fragment thereof. Thecompositions and methods of the invention include compositions andmethods for treating or preventing disorders and diseases where anincreased activity or level of NPP1 polypeptide, fragment, derivative,mutant, or mutant fragment thereof is desirable. In various embodiments,the disorders and diseases include diseases and disorders involvingpathological calcification and/or pathological ossification. Diseasesand disorders involving pathological calcification and/or pathologicalossification treatable by the compositions and methods of the invention,include, but are not limited to idiopathic infantile arterialcalcification (IIAC), ossification of the posterior longitudinalligament (OPLL), hypophosphatemic rickets, osteoarthritis, and thecalcification of atherosclerotic plaques.

In other embodiments, the invention relates to compositions and methodsfor increasing the level or activity of mutant NPP4 polypeptide orfragment thereof. The compositions and methods of the invention includecompositions and methods for treating or preventing disorders anddiseases where an increased activity or level of mutant NPP4 polypeptideor fragment thereof is desirable. In various embodiments, the disordersand diseases include diseases and disorders involving pathologicalcalcification and/or pathological ossification. Diseases and disordersinvolving pathological calcification and/or pathological ossificationtreatable by the compositions and methods of the invention, include, butare not limited to Idiopathic Infantile Arterial Calcification (IIAC),Ossification of the Posterior Longitudinal Ligament (OPLL),hypophosphatemic rickets, osteoarthritis, and calcification ofatherosclerotic plaques.

Both hemostasis and bone development are essential, finely balancedphysiologic processes that are regulated by the extracellular metabolismof purinergic signals. As described herein, to better understand therole and atomic details of purinergic signal metabolism by NPP1 andNPP4, the high-resolution structure of NPP4, the first human NPP to besolved, was determined as a way to characterize NPP4 versus NPP1structurally and enzymatically. NPP1 was shown to hydrolyze Ap3A at lownM concentrations, and either NPP1 or NPP4 in low nM concentrationspromoted irreversible platelet aggregation in human PRP in vitro.

In addition, the effect of NPP1 on platelet aggregation in the presenceof physiologic levels of Ap3A was directly measured. Despite the highdegree of sequence identity and homology, and shared structural featuresthat allow for the targeting of a mostly similar set ofnucleotide-containing substrates, these two enzymes also possess keystructural differences that account for the distinct substratespecificities central to their biological functions. NPP1 was found tobe unable to induce platelet aggregation at physiologic concentrationsreported in human blood, but could stimulate platelet aggregation iflocalized at low nM concentrations on vascular endothelium. The presentstudies describe the molecular basis of substrate discrimination by NPP4and NPP1, provide insight into their physiologic roles governing bonemineralization and platelet aggregation, and provide an apparentmechanism by which NPP1 polymorphisms associated with stroke protectionmay act.

Without wishing to be limited by any theory, the present studies suggestan alternate mechanism by which polymorphisms in NPP1 are protectiveagainst thrombotic stroke, as compared to the mechanism proposed in theprior art. The data described herein support the notion that loss offunction mutations, wherein Ap3A hydrolysis is decreased in thethrombotic microenvironment, and not gain of function mutations asproposed in the prior art, contribute to the mechanism by which thesepolymorphisms confer stroke protection. This distinction plays animportant role in designing alterations in the NPP1 and NPP4 proteinthat are useful as therapeutic agents against ectopic bonemineralization without inducing unintended side effects of apro-thrombotic state in the treated individuals. In certain embodiments,decreased NPP1 activity in brain capillaries result in decreased ADPconcentrations in the cerebral capillary bed, decreasing plateletaggregation and thrombus formation.

Further, the recognition of NPP1 as a pro-thrombotic enzyme suggeststhat recombinant enzyme replacement therapy with NPP1 or NPP4 conferssignificant thrombotic risk to patients treated with recombinant NPP1and/or NPP4 enzymes. Patients in a pro-thrombotic state are at risk forsudden death due to coronary artery and/or pulmonary artery thrombosis,and to stroke due to cerebral artery thrombosis. As demonstrated herein,the inventors have established the biochemical and physiologic rationaleto support the notion that patients treated with recombinant, unmodifiedNPP1 enzyme would be at elevated risk for these unintended effects. Therecognition by the inventors of NPP1's role in thrombosis establishesthe rationale for inducing specific point mutations in NPP1 and/or NPP4that eliminate and/or ameliorate the pro-thrombotic activity of theenzyme, while retaining the ability of the enzyme to generate PPi. Incertain embodiments, specific modification in NPP1 and NPP4 as proposedherein accomplish the goal of establishing a safe therapeutic for thetreatment of GACI and other diseases of ectopic mineralization throughthe use of recombinant NPP4 and NPP1 enzyme.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the term “Ap3P” refers toadenosine-(5′)-triphospho-(5′)-adenosine or a salt thereof.

As used herein, the term “NPP” refers to ectonucleotidepyrophosphatase/phosphodiesterase.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

A “disorder” in an animal is a state of health in which the animal isable to maintain homeostasis, but in which the animal's state of healthis less favorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a polypeptide naturally present in a living animal isnot “isolated,” but the same nucleic acid or polypeptide partially orcompletely separated from the coexisting materials of its natural stateis “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

As used herein, “substantially purified” refers to being essentiallyfree of other components. For example, a substantially purifiedpolypeptide is a polypeptide which has been separated from othercomponents with which it is normally associated in its naturallyoccurring state.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from a subject. The biological sample may contain anybiological material suitable for detecting a mRNA, polypeptide or othermarker of a physiologic or pathologic process in a subject, and maycomprise fluid, tissue, cellular and/or non-cellular material obtainedfrom the individual.

As used herein, the term “wild-type” refers to a gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designed the “normal” or “wild-type” form of the gene. Incontrast, the term “modified” or “mutant” refers to a gene or geneproduct that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics (including altered nucleic acid sequences) whencompared to the wild-type gene or gene product.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds. Synthetic polypeptides may be synthesized, for example, using anautomated polypeptide synthesizer. As used herein, the term “protein”typically refers to large polypeptides. As used herein, the term“peptide” typically refers to short polypeptides. Conventional notationis used herein to represent polypeptide sequences: the left-hand end ofa polypeptide sequence is the amino-terminus, and the right-hand end ofa polypeptide sequence is the carboxyl-terminus.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated below:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants possess at leastabout 70% homology, at least about 80% homology, at least about 90%homology, or at least about 95% homology to the native polypeptide. Theamino acid sequence variants possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence of thenative amino acid sequence.

As used herein, the terms “conservative variation” or “conservativesubstitution” as used herein refers to the replacement of an amino acidresidue by another, biologically similar residue. Conservativevariations or substitutions are not likely to change the shape of thepeptide chain. Examples of conservative variations, or substitutions,include the replacement of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

A “nucleic acid” refers to a polynucleotide and includespoly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acidsaccording to the present invention may include any polymer or oligomerof pyrimidine and purine bases, preferably cytosine, thymine, anduracil, and adenine and guanine, respectively. (See Albert L. Lehninger,Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is hereinincorporated in its entirety for all purposes). Indeed, the presentinvention contemplates any deoxyribonucleotide, ribonucleotide orpeptide nucleic acid component, and any chemical variants thereof, suchas methylated, hydroxymethylated or glucosylated forms of these bases,and the like. The polymers or oligomers may be heterogeneous orhomogeneous in composition, and may be isolated from naturally occurringsources or may be artificially or synthetically produced. In addition,the nucleic acids may be DNA or RNA, or a mixture thereof, and may existpermanently or transitionally in single-stranded or double-strandedform, including homoduplex, heteroduplex, and hybrid states.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 25 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides includesequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ormimetics thereof which may be isolated from natural sources,recombinantly produced or artificially synthesized. A further example ofa polynucleotide of the present invention may be a peptide nucleic acid(PNA). (See U.S. Pat. No. 6,156,501 which is hereby incorporated byreference in its entirety) The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably herein. It is understood thatwhen a nucleotide sequence is represented herein by a DNA sequence(e.g., A, T, G, and C), this also includes the corresponding RNAsequence (e.g., A, U, G, C) in which “U” replaces “T.”

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, contemplated are alterationsof a wild type or synthetic gene, including but not limited to deletion,insertion, substitution of one or more nucleotides, or fusion to otherpolynucleotide sequences.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

An “allele” refers to one specific form of a genetic sequence (such as agene) within a cell, an individual or within a population, the specificform differing from other forms of the same gene in the sequence of atleast one, and frequently more than one, variant sites within thesequence of the gene. The sequences at these variant sites that differbetween different alleles are termed “variants,” “polymorphisms,” or“mutations.”

As used herein the terms “alteration,” “defect,” “variation” or“mutation” refer to a mutation in a gene in a cell that affects thefunction, activity, expression (transcription or translation) orconformation of the polypeptide it encodes. Mutations encompassed by thepresent invention can be any mutation of a gene in a cell that resultsin the enhancement or disruption of the function, activity, expressionor conformation of the encoded polypeptide, including the completeabsence of expression of the encoded protein and can include, forexample, missense and nonsense mutations, insertions, deletions,frameshifts and premature terminations. Without being so limited,mutations encompassed by the present invention may alter splicing themRNA (splice site mutation) or cause a shift in the reading frame(frameshift).

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, intracellular antibodies(“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies(scFv), heavy chain antibodies, such as camelid antibodies, syntheticantibodies, chimeric antibodies, and a humanized antibodies (Harlow etal., 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

As used herein, an “immunoassay” refers to any binding assay that usesan antibody capable of binding specifically to a target molecule todetect and quantify the target molecule.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “coding sequence,” as used herein, means a sequence of anucleic acid or its complement, or a part thereof, that can betranscribed and/or translated to produce the mRNA and/or the polypeptideor a fragment thereof. Coding sequences include exons in a genomic DNAor immature primary RNA transcripts, which are joined together by thecell's biochemical machinery to provide a mature mRNA. The anti-sensestrand is the complement of such a nucleic acid, and the coding sequencecan be deduced therefrom. In contrast, the term “non-coding sequence,”as used herein, means a sequence of a nucleic acid or its complement, ora part thereof, that is not translated into amino acid in vivo, or wheretRNA does not interact to place or attempt to place an amino acid.Non-coding sequences include both intron sequences in genomic DNA orimmature primary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, and the like.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods that depend upon binding between nucleic acids.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides; at least about 1000 nucleotides to about 1500nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about2500 nucleotides (and any integer value in between). As used herein, theterm “fragment,” as applied to a protein or peptide, refers to asubsequence of a larger protein or peptide. A “fragment” of a protein orpeptide can be at least about 20 amino acids in length; for example, atleast about 50 amino acids in length; at least about 100 amino acids inlength; at least about 200 amino acids in length; at least about 300amino acids in length; or at least about 400 amino acids in length (andany integer value in between).

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or compound of the invention in the kit for identifying oralleviating or treating the various diseases or disorders recitedherein. Optionally, or alternately, the instructional material maydescribe one or more methods of identifying or alleviating the diseasesor disorders in a cell or a tissue of a subject. The instructionalmaterial of the kit may, for example, be affixed to a container thatcontains the nucleic acid, polypeptide, and/or compound of the inventionor be shipped together with a container that contains the nucleic acid,polypeptide, and/or compound. Alternatively, the instructional materialmay be shipped separately from the container with the intention that therecipient uses the instructional material and the compoundcooperatively.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the activity and/or level of a mRNA,polypeptide, or a response in a subject compared with the activityand/or level of a mRNA, polypeptide or a response in the subject in theabsence of a treatment or compound, and/or compared with the activityand/or level of a mRNA, polypeptide, or a response in an otherwiseidentical but untreated subject. The term encompasses activating,inhibiting and/or otherwise affecting a native signal or responsethereby mediating a beneficial therapeutic response in a subject,preferably, a human.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compounduseful within the invention (alone or in combination with anotherpharmaceutical agent), to a patient, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a patient(e.g., for diagnosis or ex vivo applications), who has a disease ordisorder, a symptom of a disease or disorder or the potential to developa disease or disorder, with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve or affect the disease ordisorder, the symptoms of the disease or disorder, or the potential todevelop the disease or disorder. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the patient, individual or subject is human.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof. Suitable pharmaceutically acceptable acid additionsalts may be prepared from an inorganic acid or from an organic acid.Examples of inorganic acids include sulfate, hydrogen sulfate,hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, andphosphoric acids (including hydrogen phosphate and dihydrogenphosphate). Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts of compounds of the invention include, for example,metallic salts including alkali metal, alkaline earth metal andtransition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. All of these salts may be prepared from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, inhalational,rectal, vaginal, transdermal, intranasal, buccal, sublingual,parenteral, intrathecal, intragastrical, ophthalmic, pulmonary andtopical administration.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present high-resolution structure determination of human NPP4allowed for detailed comparative structure-function studies with NPP1.These membrane-bound cell surface enzymes are involved in the metabolismof extracellular purinergic signals as well as nucleotide re-uptake.Both ectoenzymes possess a narrow hydrophobic slot adjacent to two boundzinc ions that accounts for the targeting of nucleotide-containingsubstrates, where the nucleotide base binds within the slot andhydrolysis yields the nucleotide monophosphate as one of the products.Adenine is the most-preferred base type for both enzymes and cocrystalstructures of each with a product AMP molecule bound highlight theirfunctional similarity in that region of the binding site. Both enzymeswere found to be able to hydrolyze Ap3A, but exhibit surprisinglydifferent responses to ATP.

NPP4 and NPP1 differ drastically in their response to ATP. NPP1 readilyhydrolyzes ATP to AMP and PPi, the latter of which is a potent inhibitorof extra-osseous mineralization, and mutants of NPP1 may be involved indiseases involving bone or soft-tissue calcification outlined earlier.To yield the observed products, ATP should bind NPP1 in the sameorientation seen in the NPP1-AMP cocrystal structure. In stark contrast,NPP4 cleaves ATP only exceedingly slowly, even though it binds AMP in amanner very similar to NPP1. Superposition of the two enzymes revealskey structural differences in the area of the terminal phosphate of ATP,if bound like AMP. The energy minimized simulations of ATP-complexesreveal that NPP1 provides a favorable environment for the γ-phosphate ofATP via the presence of a tri-partite lysine claw that providesinduced-fit charge-stabilization. In the absence of substrate, twolysines lining the upper ridge of the binding pocket (Lys260 and Lys510,mouse numbering) are mobile, as is demonstrated in the NPP1-AMPproduct-complex (4GTW) where they are disordered, or in theNPP1-vanadate complex (4B56) where they exhibit high B-factors andextend into the solvent. Upon ATP substrate binding, the highly-negativeγ-phosphate should electrostatically attract these lysines, which alongwith the stationary Lys237 on the floor of the binding pocket shouldserve to effectively envelop the terminal-phosphate in positive-charge.This may also promote the hydrolysis via product-stabilization, sincePPi is even more negatively-charged. The role that these lysine residuesplay in NPP1 hydrolysis of ATP had not been previously appreciated andcame to light during the detailed structural comparisons with NPP4.

Superposition of the corresponding region of NPP4 shows it to be notablyless favorable for a similarly-bound ATP, with a local architecture thatis more open, contains fewer positively-charged residues and introducesnegatively-charged residues such as Asp335, which projects into theactive site in close proximity to the γ-phosphate of ATP.Cocrystallization attempts with a non-cleavable ATP-analogue revealed novisible binding, consistent with the observation that NPP4 provides anunfavorable environment in the region of the γ-phosphate. Similarly,cocrystallization attempts with ATP or a cleavable ATP-analogue yield anAMP-complex over the several days it takes for crystals to grow,indicating that although ATP binding is weak, it occasionally comes intoclose enough proximity to be hydrolyzed. AMP product molecule is able tobind under identical conditions, reflecting a stronger affinity.Product-inhibition is likely an intrinsic feature of NPP reactions sincethe phosphate group next to Zn1 gets converted, by definition, from aphosphodiester to a terminal phosphate that carries morenegative-charge. The present data indicate that NPP4 is unlikely tohydrolyze ATP effectively in vivo, supporting the view that NPP1 is theprimary extracellular enzyme metabolizing purinergic signals regulatingbone remodeling and extracellular calcification.

In contrast, both NPP4 and NPP1 hydrolyze Ap3A into AMP and ADP, withthe Michaelis constant of NPP1 for Ap3A some 30 fold tighter than thatof NPP4. This higher affinity is reflected in the lower concentrationsof NPP1 required to trigger platelet aggregation in identicalconcentrations of Ap3A (FIGS. 10A-10B). High concentrations of Ap3Astored in the dense granules of circulating platelets are released uponplatelet activation. Without wishing to be limited by any theory,vascular bound NPPs may contribute to cerebral platelet aggregation byidentifying NPP4 on brain vascular endothelium capable of inducingplatelet aggregation via the hydrolysis of physiologic concentrations ofAp3A. Ap3A has a significantly longer lifespan in whole blood than ADPand has long been hypothesized to aid stable thrombus formation byserving as a ‘chemically masked’ source of ADP.

The ability of NPP1 to readily hydrolyze Ap3A to ADP raises the questionof whether NPP1 may play a role in hemostasis. In the present study,NPP1 was shown to be capable of Ap3A hydrolysis at low nMconcentrations, and either NPP1 or NPP4 in low nM concentrationspromotes irreversible platelet aggregation in human PRP in vitro. Thework implies that either enzyme may contribute significantly to plateletaggregation in vivo if present at low nM concentrations in apro-thrombotic environment.

Recently, polymorphisms in NPP1 have been identified that confer strokeprotection in pediatric patients with sickle cell anemia. The presentresults suggest that polymorphisms in NPP1 protective against strokerepresent a loss of function mutation that decreases Ap3A hydrolysis inthe thrombotic microenvironment. Decreased NPP1 activity in braincapillaries would result in decreased ADP concentrations in the cerebralcapillary bed, thus providing a direct mechanism to account fordecreased platelet aggregation and thrombus formation.

The K173Q mutation in human NPP1 is not in the catalytic domain, butrather is found in the somatomedin B-2 domain near the membrane spanningregion (FIG. 2B). Loss of function mutations within the NPP1 catalyticdomain are not compatible with human survival beyond the neonatalperiod, consistent with their absence in the population screened forstroke protection. Without wishing to be limited by any theory, whileNPP1 K173Q mutations may increase NPP1 serum concentrations similarly toK121Q mutations observed in IDDM-2, the hypothesis of NPP1 gain offunction is not supported by these serum increases, especially whenviewed in light of the kinetic and aggregometry data. NPP1 serumconcentrations in the K121Q polymorphism (28 pM) are well below thoserequired for NPP1-induced activation of platelet aggregation in vitro,and the Michaelis constant of NPP1 for both ATP and Ap3A is nearly 10⁶higher than the 4 pM increase induced by the K121Q polymorphism,suggesting that the increase is unlikely to impact either systemic PPior ADP concentrations. Without wishing to be bound by any particulartheory, a possible mechanism for changes attributed to the NPP1 K173Qpolymorphism could be increased ectodomain shedding of NPP1 fromvascular endothelium, which would account for both increases in serumlevels and the loss of NPP1 activity on vascular endothelium now denudedof the protein. In summation, although the means by which a K173Qmutation impairs NPP1 catalytic activity remains obscure, the presentfindings support the notion that NPP1 polymorphisms protective againststroke are more likely to represent loss function mutations thatdecrease Ap3A hydrolysis on the endothelial surface of cerebralcapillary beds, than gain of function mutations which increase PPiconcentrations.

In some embodiments, the invention relates to compositions and methodsfor increasing the ATP hydrolytic level or activity of NPP1 polypeptide,fragment, derivative, mutant, or mutant fragment thereof. In otherembodiments, the invention relates to compositions and methods inducingand increasing the ATP hydrolytic level or activity of mutant NPP4polypeptide or fragment thereof.

In some embodiments, the invention relates to compositions and methodsfor decreasing the Ap3A hydrolytic level or activity of NPP1polypeptide, fragment, derivative, mutant, or mutant fragment thereof.In other embodiments, the invention relates to compositions and methodsfor decreasing the Ap3A hydrolytic level or activity of mutant NPP4polypeptide or fragment thereof.

The methods of the invention include methods of treating or preventingdisorders and diseases where an increased activity or level of NPP1polypeptide, fragment, derivative, mutant, or mutant fragment thereof isdesirable. Thus, in some embodiments, the compositions of the inventionrelate to activators of NPP1 polypeptide, fragment, derivative, mutant,or mutant fragment thereof. In various embodiments, the disorders anddiseases include, but are not limited to IIAC, OPLL, hypophosphatemicrickets, osteoarthritis, and the calcification of atheroscleroticplaques.

In various embodiments, the mutant NPP1 polypeptide or fragment thereofuseful within the methods of the invention has lower Ap3A hydrolyticactivity as compared to the corresponding wild-type NPP1 polypeptide orfragment thereof. In various embodiments, the mutant NPP1 polypeptide orfragment thereof useful within the methods of the invention hassubstantially the same ATP hydrolytic activity as compared to thecorresponding wild-type NPP1 polypeptide or fragment thereof. In variousembodiments, the mutant NPP1 polypeptide or fragment thereof usefulwithin the methods of the invention has lower Ap3A hydrolytic activityand substantially the same ATP hydrolytic activity as compared to thecorresponding wild-type NPP1 polypeptide or fragment thereof.

In other embodiments, the methods of the invention include methods oftreating or preventing disorders and diseases where an increasedactivity or level of mutant NPP4 polypeptide or fragment thereof isdesirable. Thus, in some embodiments, the compositions of the inventionrelate to activators of mutant NPP4 polypeptide or fragment thereof. Invarious embodiments, the disorders and diseases include, but are notlimited to IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, andcalcification of atherosclerotic plaques. In some embodiments, theinvention relates to compositions and methods for altering the enzymaticactivity of mutant NPP4 polypeptide or fragment thereof into ahydrolase, or an enzyme with the same enzymatic activity of NPP1polypeptide, fragment, derivative, mutant, or mutant fragment thereof.

In various embodiments, the mutant NPP4 polypeptide or fragment thereofuseful within the methods of the invention has lower Ap3A hydrolyticactivity as compared to the corresponding wild-type NPP4 polypeptide orfragment thereof. In other embodiments, the mutant NPP4 polypeptide orfragment thereof useful within the methods of the invention hassubstantially increased ATP hydrolytic activity as compared to thecorresponding wild-type NPP4 polypeptide or fragment thereof. In yetother embodiments, the mutant NPP4 polypeptide or fragment thereofuseful within the methods of the invention has lower Ap3A hydrolyticactivity and substantially increased ATP hydrolytic activity as comparedto the corresponding wild-type NPP4 polypeptide or fragment thereof.

In further embodiments, the invention relates to compositions andmethods for increasing the level or activity of NPP1 polypeptide,fragment, derivative, mutant, or mutant fragment thereof, and mutantNPP4 polypeptide or fragment thereof.

NPP1 Therapeutic Activator Compositions and Methods

The present invention includes NPP1 activator compositions and methodsof increasing the level or activity of NPP1 or a mutant thereof. Invarious embodiments, the NPP1 activator compositions and methods oftreatment of the invention increase the amount of NPP1 polypeptide, NPP1mRNA, NPP1 enzymatic activity, NPP1 substrate binding activity, a mutantthereof or a combination thereof. In various embodiments, the diseasesand disorders where a decrease in pathological calcification orossification may improve therapeutic outcome include, but are notlimited to, IIAC, OPLL, hypophosphatemic rickets, osteoarthritis, andcalcification of atherosclerotic plaques.

In various embodiments, the mutant NPP1 useful within the methods of theinvention has lower Ap3A hydrolytic activity as compared to thecorresponding wild-type NPP1. In various embodiments, the mutant NPP1useful within the methods of the invention has substantially the sameATP hydrolytic activity as compared to the corresponding wild-type NPP1.In various embodiments, the mutant NPP1 useful within the methods of theinvention has lower Ap3A hydrolytic activity and substantially the sameATP hydrolytic activity as compared to the corresponding wild-type NPP1.In various embodiments, the mutant NPP1 has a mutation in at least oneposition selected from the group consisting of Ser 532, Tyr 529, Tyr451, Ile 450, Ser 381, Tyr 382, Ser 377, Phe 346, Gly 531, Ser 289, Ser287, Ala 454, Gly 452, Gln 519, Glu 526, Lys 448, Glu 508, Arg 456, Asp276, Tyr 434, Gln 519, Ser 525, Gly 342, Ser 343 and Gly 536. Withoutwishing to be limited by any theory, the mutations contemplated withinthe invention are informed by the high-resolution structuredetermination of NPP4 by the inventors, and the correct interpretationof the lysine claw in NPP1 that facilitates ATP hydrolysis by NPP1(FIGS. 6A-6C, 7A-7D, 7, 9A-9F).

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of NPP1 ormutant thereof encompasses the increase in expression, includingtranscription, translation, or both, of NPP1 or mutant thereof. Theskilled artisan will also appreciate, once armed with the teachings ofthe present invention, that an increase in the level of NPP1 or mutantthereof includes an increase in activity (e.g., enzymatic activity,substrate binding activity, etc.) of NPP1 or mutant thereof. Thus,increasing the level or activity of NPP1 or mutant thereof includes, butis not limited to, increasing the amount of NPP1 polypeptide or mutantthereof, and increasing transcription, translation, or both, of anucleic acid encoding NPP1 or mutant thereof; and it also includesincreasing any activity of an NPP1 polypeptide or mutant thereof aswell. The compositions and methods of the invention can selectivelyactivate NPP1 or mutant thereof, or can activate both NPP1 or mutantthereof and another molecule, such as, by way of a non-limiting example,mutant NPP4.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, including the three-dimensional structurereported herein, that an increase in the level of NPP1 activity ormutant thereof encompasses the manipulation of specific residues in NPP1to effect alterations of the Michaelis-Menton constants to eitherincrease the affinity of the enzyme for ATP (K_(m)) or to increase theturnover rate of ATP into PP_(i) by NPP1 (k_(cat)). The skilled artisanwill also appreciate, once armed with the teachings of the presentinvention, and the three-dimensional structure reported herein, thatthis may be accomplished through the substitution of residues within theactive site of NPP1, as enabled by disclosures, findings, and knowledgecontained and described herein. Mutations of this effect are thereforeclaimed as methods of art by this application.

The skilled artisan will also appreciate, once armed with the teachingsof the present invention, and the three-dimensional structure reportedherein, that decreasing the pro-thrombotic activity of NPP1 or a mutantthereof encompasses the manipulation of specific residues in NPP1 toeffect alterations of the Michaelis-Menton constants of the enzyme todecrease the affinity of NPP1 for Ap3A (K_(m)), or to decrease theturnover rate of Ap3A into ADP by NPP1 (k_(cat)). Mutations whichaccomplish this effect are therefore claimed as methods of art by thisapplication. The skilled artisan will also appreciate, once armed withthe teachings of the present invention, and the three-dimensionalstructure reported herein, including the docked molecule of Ap3A intoNPP1 detailed in FIGS. 9A-9F and 11, that this may be accomplishedthrough the substitution or alterations of amino acids lining theadenine binding pockets of NPP4 into amino acids that increase theoccupied space of the amino acids which reduce or eliminate the adeninebinding pocket of Ap3A, as enabled by disclosures, findings, themolecular structure of NPP1, and the use of the molecular structures ofthe model of NPP1 bound to Ap3A as depicted in FIGS. 6A-6C-7A-7D, 9A-9F,and 11, and other knowledge contained and described herein. Mutations tothis effect are therefore claimed as methods of art by this application.

Thus, the present invention relates to the prevention and treatment of adisease or disorder by administration of an NPP1 polypeptide, arecombinant NPP1 polypeptide, a mutant NPP1 polypeptide, an active NPP1polypeptide fragment, or an activator of NPP1 expression or activity. Inone embodiment, the NPP1 polypeptide or mutant thereof is soluble. Inanother embodiment, the NPP1 polypeptide or mutant thereof is arecombinant NPP1 polypeptide. In one embodiment, the NPP1 polypeptide ormutant thereof includes an NPP1 polypeptide that lacks the NPP1transmembrane domain. In another embodiment, the NPP1 polypeptide ormutant thereof includes an NPP1 polypeptide where the NPP1 transmembranedomain has been removed and replaced with the transmembrane domain ofanother polypeptide, such as, by way of non-limiting example, NPP2.

In some embodiments, the NPP1 polypeptide or mutant thereof comprises anIgG Fc domain. In other embodiments, the NPP1 polypeptide or mutantthereof comprises a polyaspartic acid domain comprise from about 2 toabout 20 or more sequential aspartic acid residues to target the NPP1polypeptide to bone. In some embodiments, the NPP1 polypeptide or mutantthereof comprises an IgG Fc domain and a polyaspartic acid domaincomprising from about 2 to about 20 or more sequential aspartic acidresidues. In other embodiments, the NPP1 protein or mutant thereof istruncated to remove the nuclease domain. In a particular embodiment, theNPP1 protein or mutant thereof is truncated to remove the nucleasedomain from about residue 524 to about residue 885 relative to SEQ IDNO:1, leaving only the catalytic domain from about residue 186 to aboutresidue 586 relative to SEQ ID NO:1, which serves to preserve thecatalytic activity of the protein.

It is understood by one skilled in the art, that an increase in thelevel of NPP1 or mutant thereof encompasses an increase in the amount ofNPP1 or mutant thereof (e.g., by administration of NPP1, fragmentthereof or mutant thereof, by increasing NPP1 protein expression, etc.).Additionally, the skilled artisan would appreciate, that an increase inthe level of NPP1 or mutant thereof includes an increase in NPP1activity. Thus, increasing the level or activity of NPP1 or mutantthereof includes, but is not limited to, the administration of NPP1,fragment thereof or mutant thereof, as well as increasing transcription,translation, or both, of a nucleic acid encoding NPP1 or mutant thereof;and it also includes increasing any activity of NPP1 or mutant thereofas well.

The increased level or activity of NPP1 or mutant thereof can beassessed using a wide variety of methods, including those disclosedherein, as well as methods well-known in the art or to be developed inthe future. That is, the routineer would appreciate, based upon thedisclosure provided herein, that increasing the level or activity ofNPP1 or mutant thereof can be readily assessed using methods that assessthe level of a nucleic acid encoding NPP1 or mutant thereof (e.g., mRNA)to alter the Michaelis Menton kinetics to substrate and product turnoveras described herein, the level of NPP1 polypeptide or mutant thereof,and/or the level of activity in a biological sample obtained from asubject.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in subjects who, in whole (e.g.,systemically) or in part (e.g., locally, tissue, organ), are being, orwill be, treated for pathological calcification or ossification. In oneembodiment, the invention is useful in treating or preventingpathological calcification or ossification. The skilled artisan willappreciate, based upon the teachings provided herein, that the diseasesand disorders treatable by the compositions and methods described hereinencompass any disease or disorder where a decrease in calcification orossification will promote a positive therapeutic outcome.

One of skill in the art will realize that in addition to activating NPP1or mutant thereof directly, diminishing the amount or activity of amolecule that itself diminishes the amount or activity of NPP1 or mutantthereof can serve to increase the amount or activity of NPP1 or mutantthereof. Thus, an activator can include, but should not be construed asbeing limited to, a chemical compound, a protein, a peptidomimetic, anantibody, a ribozyme, and an antisense nucleic acid molecule. One ofskill in the art would readily appreciate, based on the disclosureprovided herein, that an activator encompasses a chemical compound thatincreases the level, enzymatic activity, or substrate binding activityof NPP1 or mutant thereof. Additionally, an activator encompasses achemically modified compound, and derivatives, as is well known to oneof skill in the chemical arts.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of NPP1 ormutant thereof encompasses the increase in expression, includingtranscription, translation, or both, of NPP1 or mutant thereof. Theskilled artisan will also appreciate, once armed with the teachings ofthe present invention, that an increase in the level of NPP1 or mutantthereof includes an increase in activity (e.g., enzymatic activity,substrate binding activity, etc.) of NPP1 or mutant thereof. Thus,increasing the level or activity of NPP1 or mutant thereof includes, butis not limited to, increasing the amount of NPP1 polypeptide or mutantthereof, increasing transcription, translation, or both, of a nucleicacid encoding NPP1 or mutant thereof; and it also includes increasingany activity of an NPP1 polypeptide or mutant thereof as well. Theactivator compositions and methods of the invention can selectivelyactivate NPP1 or mutant thereof, or can activate both NPP1 or mutantthereof and another molecule, such as, by way of non-limiting example,NPP4.

Thus, the present invention relates to administration of an NPP1polypeptide, a recombinant NPP1 polypeptide, a mutant NPP1 polypeptide,an active NPP1 polypeptide fragment, or an activator of NPP1 expressionor activity. In one embodiment, the NPP1 polypeptide or mutant thereofis soluble. In another embodiment, the NPP1 polypeptide or mutantthereof is a recombinant polypeptide. In one embodiment, the NPP1polypeptide or mutant thereof includes an NPP1 polypeptide or mutantthereof that lacks the NPP1 transmembrane domain. In another embodiment,the NPP1 polypeptide or mutant thereof includes an NPP1 polypeptide ormutant thereof where the NPP1 transmembrane domain or mutant thereof hasbeen removed and replaced with the transmembrane domain of anotherpolypeptide, such as, by way of non-limiting example, NPP2.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that an NPP1activator includes such activators as discovered in the future, as canbe identified by well-known criteria in the art of pharmacology, such asthe physiological results of activation of NPP1 as described in detailherein and/or as known in the art. Therefore, the present invention isnot limited in any way to any particular NPP1 activator or mutant NPP1activator as exemplified or disclosed herein; rather, the inventionencompasses those activators that would be understood by the routineerto be useful as are known in the art and as are discovered in thefuture.

Further methods of identifying and producing an NPP1 activator are wellknown to those of ordinary skill in the art, including, but not limited,obtaining an activator from a naturally occurring source (e.g.,Streptomyces sp Pseudomonas sp., Stylotella aurantium, etc.).Alternatively, an NPP1 activator can be synthesized chemically. Further,the routineer would appreciate, based upon the teachings providedherein, that an NPP1 activator can be obtained from a recombinantprotein expression system, including but not limited to mammalianprotein expression systems, insect cell protein expression systems, andyeast protein expression systems. Compositions and methods forchemically synthesizing NPP1 activators or mutant NPP1 activators andfor obtaining them from natural sources are well known in the art andare described in the art.

One of skill in the art will appreciate that an activator can beadministered as a small molecule chemical, a protein, a nucleic acidconstruct encoding a protein, or combinations thereof. Numerous vectorsand other compositions and methods are well known for administering aprotein or a nucleic acid construct encoding a protein to cells ortissues. Therefore, the invention includes a method of administering aprotein or a nucleic acid encoding a protein that is an activator ofNPP1 or mutant thereof (Sambrook et al., 2012, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel etal., 1997, Current Protocols in Molecular Biology, John Wiley & Sons,New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself diminishes the amount or activity ofNPP1 or mutant thereof can serve to increase the amount or activity ofNPP1 or mutant thereof. Antisense oligonucleotides are DNA or RNAmolecules that are complementary to some portion of an mRNA molecule.When present in a cell, antisense oligonucleotides hybridize to anexisting mRNA molecule and inhibit translation into a gene product.Inhibiting the expression of a gene using an antisense oligonucleotideis well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289),as are methods of expressing an antisense oligonucleotide in a cell(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention includethe use of antisense oligonucleotide to diminish the amount of amolecule that causes a decrease in the amount or activity NPP1 or mutantthereof, thereby increasing the amount or activity of NPP1 or mutantthereof. Contemplated in the present invention are antisenseoligonucleotides that are synthesized and provided to the cell by way ofmethods well known to those of ordinary skill in the art. As an example,an antisense oligonucleotide can be synthesized to be between about 10and about 100, more preferably between about 15 and about 50 nucleotideslong. The synthesis of nucleic acid molecules is well known in the art,as is the synthesis of modified antisense oligonucleotides to improvebiological activity in comparison to unmodified antisenseoligonucleotides (U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing a protein that diminishesthe level or activity of NPP1 or mutant thereof can be accomplishedthrough the use of a ribozyme. Using ribozymes for inhibiting geneexpression is well known to those of skill in the art (see, e.g., Cechet al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989,Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).Ribozymes are catalytic RNA molecules with the ability to cleave othersingle-stranded RNA molecules. Ribozymes are known to be sequencespecific, and can therefore be modified to recognize a specificnucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowingthe selective cleavage of specific mRNA molecules. Given the nucleotidesequence of the molecule, one of ordinary skill in the art couldsynthesize an antisense oligonucleotide or ribozyme without undueexperimentation, provided with the disclosure and referencesincorporated herein.

One of skill in the art will appreciate that an NPP1 activator, NPP1polypeptide, a recombinant NPP1 polypeptide, a mutant NPP1 polypeptide,or an active NPP1 polypeptide fragment can be administered singly or inany combination thereof. One of skill in the art will also appreciateadministration can be acute (e.g., over a short period of time, such asa day, a week or a month) or chronic (e.g., over a long period of time,such as several weeks, several months or a year or more). Further, anNPP1 polypeptide, a recombinant NPP1 polypeptide, a mutant NPP1polypeptide, or an active NPP1 polypeptide fragment can be administeredsingly or in any combination thereof in a temporal sense, in that theymay be administered simultaneously, before, and/or after each other. Oneof ordinary skill in the art will appreciate, based on the disclosureprovided herein, that an NPP1 polypeptide, a recombinant NPP1polypeptide, a mutant NPP1 polypeptide, or an active NPP1 polypeptidefragment can be used to treat or prevent pathological calcification orossification, and that an activator can be used alone or in anycombination with another NPP1 polypeptide, recombinant NPP1 polypeptide,active NPP1 polypeptide fragment, or NPP1 activator to effect atherapeutic result.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder once isestablished. Particularly, the symptoms of the disease or disorder neednot have manifested to the point of detriment to the subject; indeed,the disease or disorder need not be detected in a subject beforetreatment is administered. That is, significant pathology from diseaseor disorder does not have to occur before the present invention mayprovide benefit. Therefore, the present invention, as described morefully herein, includes a method for preventing diseases and disorders ina subject, in that an NPP1 polypeptide, fragment, derivative or mutantthereof, or an NPP1 activator or mutant NPP1 activator, as discussedelsewhere herein, can be administered to a subject prior to the onset ofthe disease or disorder, thereby preventing the disease or disorder fromdeveloping.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of a disease or disorder in a subjectencompasses administering to a subject an NPP1 polypeptide, arecombinant NPP1 polypeptide, a mutant NPP1 polypeptide, an active NPP1polypeptide fragment, or NPP1 activator as a preventative measureagainst a disease or disorder. In one embodiment, the NPP1 polypeptideis soluble. In another embodiment, the NPP1 polypeptide is a recombinantNPP1 polypeptide. In one embodiment, the NPP1 polypeptide includes anNPP1 polypeptide that lacks the NPP1 transmembrane domain. In anotherembodiment, the NPP1 polypeptide includes an NPP1 polypeptide where theNPP1 transmembrane domain has been removed and replaced with thetransmembrane domain of another polypeptide, such as, by way ofnon-limiting example, NPP2.

In some embodiments, the NPP1 polypeptide comprises an IgG Fc domain. Inother embodiments, the NPP1 polypeptide comprises a polyaspartic aciddomain comprise from about 2 to about 20 or more sequential asparticacid residues to target the NPP1 polypeptide to bone. In someembodiments, the NPP1 polypeptide comprises an IgG Fc domain and apolyaspartic acid domain comprising from about 2 to about 20 or moresequential aspartic acid residues. In other embodiments, the NPP1protein is truncated to remove the nuclease domain. In a particularembodiment, NPP1 protein is truncated to remove the nuclease domain fromabout residue 524 to about residue 885 relative to SEQ ID NO:1, leavingonly the catalytic domain from about residue 186 to about residue 586relative to SEQ ID NO:1, which serves to preserve the catalytic activityof the protein.

As more fully discussed elsewhere herein, methods of increasing thelevel or activity of an NPP1 encompass a wide plethora of techniques forincreasing not only NPP1 activity, but also for increasing expression ofa nucleic acid encoding NPP1. Additionally, as disclosed elsewhereherein, one skilled in the art would understand, once armed with theteaching provided herein, that the present invention encompasses amethod of preventing a wide variety of diseases or disorders whereincreased expression and/or activity of NPP1 mediates, treats orprevents a disease or disorder. Further, the invention encompassestreatment or prevention of such diseases or disorders discovered in thefuture.

The invention encompasses administration of an NPP1 polypeptide, arecombinant NPP1 polypeptide, a mutant NPP1 polypeptide, an active NPP1polypeptide fragment, an NPP1 activator, or a mutant NPP4 polypeptidemodified to exhibit NPP1-like ATP hydrolase activity to practice themethods of the invention; the skilled artisan would understand, based onthe disclosure provided herein, how to formulate and administer theappropriate NPP1 polypeptide, recombinant NPP1 polypeptide, active NPP1polypeptide fragment, or NPP1 activator to a subject. However, thepresent invention is not limited to any particular method ofadministration or treatment regimen. This is especially true where itwould be appreciated by one skilled in the art, equipped with thedisclosure provided herein, including the reduction to practice using anart-recognized model of pathological calcification or ossification, thatmethods of administering an NPP1 polypeptide, a recombinant NPP1polypeptide, a mutant NPP1 polypeptide, an active NPP1 polypeptidefragment, or NPP1 activator can be determined by one of skill in thepharmacological arts.

NPP4 Therapeutic Activator Compositions and Methods

In various embodiments, the present invention includes NPP4 activatorcompositions and methods of increasing the level or activity of NPP4. Invarious embodiments, the NPP4 activator compositions and methods oftreatment of the invention increase the amount of NPP4 polypeptide, theamount of NPP4 mRNA, the amount of NPP4 enzymatic activity, the amountof NPP4 substrate binding activity, or a combination thereof. In variousembodiments, the diseases and disorders where a decrease in pathologicalcalcification or ossification may improve therapeutic outcome include,but are not limited to, IIAC, OPLL, hypophosphatemic rickets,osteoarthritis, and the calcification of atherosclerotic plaques.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, including the three-dimensional structurereported herein, that an increase in the ATP hydrolytic activity of NPP4or mutant thereof encompasses manipulations of specific amino acids inNPP4 to affect alteration of the Michaelis-Menton constants that eitherincrease the affinity of the enzyme for ATP (increase the K_(m)) or toincrease the turnover rate of ATP into PP_(i) by NPP4 (increase thek_(cat)). The skilled artisan will also appreciate, once armed with theteachings of the present invention, including the three-dimensionalstructure reported herein, that this may be accomplished through thesubstitution of residues within the active site of NPP1 into the activesite of NPP4, as enabled by disclosures, findings, and knowledgecontained and described herein, including but not limited to thedetailed analysis of the active sites of the two enzyme depicted inFIGS. 9A-9F, and the reproduction of the lysine claw in NPP1 withinNPP4. Mutations of this effect are therefore claimed as methods of artby this application.

The skilled artisan will also appreciate, once armed with the teachingsof the present invention, and the three-dimensional structure reportedherein, that decreasing the pro-thrombotic activity of NPP4 or a mutantthereof encompasses the alteration of the Michaelis-Menton constants ofthe enzyme to decrease the affinity of NPP4 for Ap3A (decrease theK_(m)), or to decrease the turnover rate of Ap3A into ADP by NPP1(decrease the k_(cat)). The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, and thethree-dimensional structure reported herein, including the dockedmolecule of Ap3A into NPP4, that this may be accomplished through thesubstitution or alterations of amino acids lining the adenine bindingpockets of NPP4 into amino acids that increase the occupied space ofsaid amino acids to reduce or eliminate the adenine binding pockets ofAp3A within NPP4, as enabled by disclosures, findings, the molecularstructure of NPP4, and the molecular structure of the model of NPP4bound to Ap3A, as detailed in FIGS. 6A-6C,-7A-7D, 9A-9F and 11, andother knowledge contained and described herein. Mutations of this effectare therefore claimed as methods of art by this application.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of NPP4encompasses the increase in NPP4 expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that an increase inthe level of NPP4 includes an increase in NPP4 activity (e.g., enzymaticactivity, substrate binding activity, etc.). Thus, increasing the levelor activity of NPP4 includes, but is not limited to, increasing theamount of NPP4 polypeptide, and increasing transcription, translation,or both, of a nucleic acid encoding NPP4; and it also includesincreasing any activity of an NPP4 polypeptide as well. The NPP4activator compositions and methods of the invention can selectivelyactivate NPP4, or can activate both NPP4 and another molecule, such as,by way of a non-limiting example, NPP1.

Thus, the present invention relates to the prevention and treatment of adisease or disorder by administration of NPP4, including an NPP4polypeptide, a recombinant NPP4 polypeptide, a mutant NPP4 polypeptide,an active NPP4 polypeptide fragment, or an activator of NPP4 expressionor activity. In one embodiment, the NPP4 is soluble. In anotherembodiment, the NPP4 is a recombinant NPP4 polypeptide. In oneembodiment, the NPP4 includes an NPP4 polypeptide that lacks the NPP4transmembrane domain. In another embodiment, the NPP4 includes an NPP4polypeptide where the NPP4 transmembrane domain has been removed andreplaced with the transmembrane domain of another polypeptide. In oneembodiment, the NPP4 includes an NPP4 polypeptide that lacks the NPP4cytoplasmic domain. In another embodiment, the NPP4 includes an NPP4polypeptide where the NPP4 cytoplasmic domain has been removed andreplaced with the cytoplasmic domain of another polypeptide. In yetanother embodiment, the NPP4 is modified to exhibit NPP1-like ATPhydrolytic activity. In one embodiment, the mutant NPP4 polypeptide ismodified to exhibit NPP1-like ATP hydrolytic activity is fused to IgG Fcand/or a polyaspartic acid domains, as described elsewhere herein. Inone embodiment, the mutant NPP4 polypeptide comprises at least onemutation that changes the substrate selectivity of the NPP4 polypeptidefrom Ap3A to ATP. In various embodiments, the mutant NPP4 polypeptidecomprises at least one mutation that changes the substrate selectivityof the NPP4 polypeptide from Ap3A to ATP, such as, by way ofnon-limiting examples, D335, S92, D264, L265, 5330, Q331, K332, or T323,relative to SEQ ID NO:3.

It is understood by one skilled in the art, that an increase in thelevel of NPP4 encompasses an increase in the amount of NPP4 (e.g., byadministration of NPP4 or a mutant or fragment thereof, by increasingNPP4 protein expression, etc.). Additionally, the skilled artisan wouldappreciate, that an increase in the level of NPP4 includes an increasein NPP4 activity. Thus, increasing the level or activity of NPP4includes, but is not limited to, the administration of NPP4 or a mutantor fragment thereof, as well as increasing transcription, translation,or both, of a nucleic acid encoding NPP4; and it also includesincreasing any activity of NPP4 as well.

The increased level or activity of NPP4 can be assessed using a widevariety of methods, including those disclosed herein, as well as methodswell-known in the art or to be developed in the future. That is, theroutineer would appreciate, based upon the disclosure provided herein,that increasing the level or activity of NPP4 can be readily assessedusing methods that assess the level of a nucleic acid encoding NPP4(e.g., mRNA), the level of NPP4 polypeptide, and/or the level of NPP4activity in a biological sample obtained from a subject.

One skilled in the art, based upon the disclosure provided herein, wouldunderstand that the invention is useful in subjects who, in whole (e.g.,systemically) or in part (e.g., locally, tissue, organ), are being orwill be, treated for pathological calcification or ossification. In oneembodiment, the invention is useful in treating or preventingpathological calcification or ossification. The skilled artisan willappreciate, based upon the teachings provided herein, that the diseasesand disorders treatable by the compositions and methods described hereinencompass any disease or disorder where a decrease in calcification orossification will promote a positive therapeutic outcome.

One of skill in the art will realize that in addition to activating NPP4directly, diminishing the amount or activity of a molecule that itselfdiminishes the amount or activity of NPP4 can serve to increase theamount or activity of NPP4. Thus, an NPP4 activator can include, butshould not be construed as being limited to, a chemical compound, aprotein, a peptidomimetic, an antibody, a ribozyme, and an antisensenucleic acid molecule. One of skill in the art would readily appreciate,based on the disclosure provided herein, that an NPP4 activatorencompasses a chemical compound that increases the level, enzymaticactivity, or substrate binding activity of NPP4. Additionally, an NPP4activator encompasses a chemically modified compound, and derivatives,as is well known to one of skill in the chemical arts.

It will be understood by one skilled in the art, based upon thedisclosure provided herein, that an increase in the level of NPP4encompasses the increase in NPP4 expression, including transcription,translation, or both. The skilled artisan will also appreciate, oncearmed with the teachings of the present invention, that an increase inthe level of NPP4 includes an increase in NPP4 activity (e.g., enzymaticactivity, substrate binding activity, etc.). Thus, increasing the levelor activity of NPP4 includes, but is not limited to, increasing theamount of NPP4 polypeptide, increasing transcription, translation, orboth, of a nucleic acid encoding NPP4; and it also includes increasingany activity of an NPP4 polypeptide as well. The NPP4 activatorcompositions and methods of the invention can selectively activate NPP4,or can activate both NPP4 and another molecule, such as, by way ofnon-limiting example, NPP1. Thus, the present invention relates toadministration of an NPP4 polypeptide, a recombinant NPP4 polypeptide, amutant NPP4 polypeptide, an active NPP4 polypeptide fragment, or anactivator of NPP4 expression or activity. In one embodiment, the NPP4 issoluble. In another embodiment, the NPP4 is a recombinant NPP4polypeptide. In one embodiment, the NPP4 includes an NPP4 polypeptidethat lacks the NPP4 transmembrane domain. In another embodiment, theNPP4 includes an NPP4 polypeptide where the NPP4 transmembrane domainhas been removed and replaced with the transmembrane domain of anotherpolypeptide. In one embodiment, the NPP4 includes an NPP4 polypeptidethat lacks the NPP4 cytoplasmic domain. In another embodiment, the NPP4includes an NPP4 polypeptide where the NPP4 cytoplasmic domain has beenremoved and replaced with the cytoplasmic domain of another polypeptide.In yet another embodiment, the NPP4 is modified to exhibit NPP1-like ATPhydrolytic activity. In one embodiment, the NPP4 modified to exhibitNPP1-like ATP hydrolytic activity is fused to IgG Fc and/or polyasparticacid domains, as described elsewhere herein. In one embodiment, themutant NPP4 polypeptide comprises at least one mutation that changes thesubstrate selectivity of the NPP4 polypeptide from Ap3A to ATP. Invarious embodiments, the NPP4 polypeptide comprises at least onemutation that changes the substrate selectivity of the NPP4 polypeptidefrom Ap3A to ATP, such as, by way of non-limiting examples, D335, S92,D264, L265, 5330, Q331, K332, or T323, relative to SEQ ID NO:3.

Further, one of skill in the art would, when equipped with thisdisclosure and the methods exemplified herein, appreciate that an NPP4activator includes such activators as discovered in the future, as canbe identified by well-known criteria in the art of pharmacology, such asthe physiological results of activation of NPP4 as described in detailherein and/or as known in the art. Therefore, the present invention isnot limited in any way to any particular NPP4 activator as exemplifiedor disclosed herein; rather, the invention encompasses those activatorsthat would be understood by the routineer to be useful as are known inthe art and as are discovered in the future.

Further methods of identifying and producing an NPP4 activator are wellknown to those of ordinary skill in the art, including, but not limited,obtaining an activator from a naturally occurring source (e.g.,Streptomyces sp., Pseudomonas sp., Stylotella aurantium, etc.).Alternatively, an NPP4 activator can be synthesized chemically. Further,the routineer would appreciate, based upon the teachings providedherein, that an NPP4 activator can be obtained from a recombinantorganism. Compositions and methods for chemically synthesizing NPP4activators and for obtaining them from natural sources are well known inthe art and are described in the art.

One of skill in the art will appreciate that an activator can beadministered as a small molecule chemical, a protein, a nucleic acidconstruct encoding a protein, or combinations thereof. Numerous vectorsand other compositions and methods are well known for administering aprotein or a nucleic acid construct encoding a protein to cells ortissues. Therefore, the invention includes a method of administering aprotein or a nucleic acid encoding a protein that is an activator ofNPP4 (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York).

One of skill in the art will realize that diminishing the amount oractivity of a molecule that itself diminishes the amount or activity ofNPP4 can serve to increase the amount or activity of NPP4. Antisenseoligonucleotides are DNA or RNA molecules that are complementary to someportion of an mRNA molecule. When present in a cell, antisenseoligonucleotides hybridize to an existing mRNA molecule and inhibittranslation into a gene product. Inhibiting the expression of a geneusing an antisense oligonucleotide is well known in the art(Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods ofexpressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.5,190,931). The methods of the invention include the use of antisenseoligonucleotide to diminish the amount of a molecule that causes adecrease in the amount or activity NPP4, thereby increasing the amountor activity of NPP4. Contemplated in the present invention are antisenseoligonucleotides that are synthesized and provided to the cell by way ofmethods well known to those of ordinary skill in the art. As an example,an antisense oligonucleotide can be synthesized to be between about 10and about 100, more preferably between about 15 and about 50 nucleotideslong. The synthesis of nucleic acid molecules is well known in the art,as is the synthesis of modified antisense oligonucleotides to improvebiological activity in comparison to unmodified antisenseoligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,243).

Similarly, the expression of a gene may be inhibited by thehybridization of an antisense molecule to a promoter or other regulatoryelement of a gene, thereby affecting the transcription of the gene.Methods for the identification of a promoter or other regulatory elementthat interacts with a gene of interest are well known in the art, andinclude such methods as the yeast two hybrid system (Bartel and Fields,eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary,N.C.).

Alternatively, inhibition of a gene expressing a protein that diminishesthe level or activity of NPP4 can be accomplished through the use of aribozyme. Using ribozymes for inhibiting gene expression is well knownto those of skill in the art (see, e.g., Cech et al., 1992, J. Biol.Chem. 267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman etal., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA moleculeswith the ability to cleave other single-stranded RNA molecules.Ribozymes are known to be sequence specific, and can therefore bemodified to recognize a specific nucleotide sequence (Cech, 1988, J.Amer. Med. Assn. 260:3030), allowing the selective cleavage of specificmRNA molecules. Given the nucleotide sequence of the molecule, one ofordinary skill in the art could synthesize an antisense oligonucleotideor ribozyme without undue experimentation, provided with the disclosureand references incorporated herein.

One of skill in the art will appreciate that an NPP4 activator, NPP4polypeptide, a recombinant NPP4 polypeptide, a mutant NPP4 polypeptide,or an active NPP4 polypeptide fragment can be administered singly or inany combination thereof. One of skill in the art will also appreciateadministration can be acute (e.g., over a short period of time, such asa day, a week or a month) or chronic (e.g., over a long period of time,such as several weeks, several months or a year or more). Further, anNPP4 polypeptide, a recombinant NPP4 polypeptide, a mutant NPP4polypeptide, or an active NPP4 polypeptide fragment can be administeredsingly or in any combination thereof in a temporal sense, in that theymay be administered simultaneously, before, and/or after each other. Oneof ordinary skill in the art will appreciate, based on the disclosureprovided herein, that an NPP4 polypeptide, a recombinant NPP4polypeptide, a mutant NPP4 polypeptide, or an active NPP4 polypeptidefragment can be used to treat or prevent pathological calcification orossification, and that an activator can be used alone or in anycombination with another NPP4 polypeptide, recombinant NPP4 polypeptide,active NPP4 polypeptide fragment, or NPP4 activator to effect atherapeutic result.

It will be appreciated by one of skill in the art, when armed with thepresent disclosure including the methods detailed herein, that theinvention is not limited to treatment of a disease or disorder once isestablished. Particularly, the symptoms of the disease or disorder neednot have manifested to the point of detriment to the subject; indeed,the disease or disorder need not be detected in a subject beforetreatment is administered. That is, significant pathology from diseaseor disorder does not have to occur before the present invention mayprovide benefit. Therefore, the present invention, as described morefully herein, includes a method for preventing diseases and disorders ina subject, in that an NPP4 polypeptide, or a fragment, derivative, ormutant thereof, or an NPP4 activator, as discussed elsewhere herein, canbe administered to a subject prior to the onset of the disease ordisorder, thereby preventing the disease or disorder from developing.

One of skill in the art, when armed with the disclosure herein, wouldappreciate that the prevention of a disease or disorder in a subjectencompasses administering to a subject NPP4, including an NPP4polypeptide, a recombinant NPP4 polypeptide, a mutant NPP4 polypeptide,an active NPP4 polypeptide fragment, or NPP4 activator as a preventativemeasure against a disease or disorder. In one embodiment, the NPP4 issoluble. In another embodiment, the NPP4 is a recombinant NPP4polypeptide. In one embodiment, the NPP4 includes an NPP4 polypeptidethat lacks the NPP4 transmembrane domain. In another embodiment, theNPP4 includes an NPP4 polypeptide where the NPP4 transmembrane domainhas been removed and replaced with the transmembrane domain of anotherpolypeptide. In one embodiment, the NPP4 includes an NPP4 polypeptidethat lacks the NPP4 cytoplasmic domain. In another embodiment, the NPP4includes an NPP4 polypeptide where the NPP4 cytoplasmic domain has beenremoved and replaced with the cytoplasmic domain of another polypeptide.In yet another embodiment, the NPP4 is modified to exhibit NPP1-like ATPhydrolytic activity. In one embodiment, the NPP4 modified to exhibitNPP1-like ATP hydrolytic activity is fused to IgG Fc and/or polyasparticacid domains, as described elsewhere herein. In one embodiment, themutant NPP4 polypeptide comprises at least one mutation that changes thesubstrate selectivity of the NPP4 polypeptide from Ap3A to ATP. Invarious embodiments, the mutant NPP4 polypeptide comprises at least onemutation that changes the substrate selectivity of the NPP4 polypeptidefrom Ap3A to ATP, such as, by way of non-limiting examples, D335, S92,D264, L265, 5330, Q331, K332, or T323, relative to SEQ ID NO:3.

As more fully discussed elsewhere herein, methods of increasing thelevel or activity of an NPP4 encompass a wide plethora of techniques forincreasing not only NPP4 activity, but also for increasing expression ofa nucleic acid encoding NPP4. Additionally, as disclosed elsewhereherein, one skilled in the art would understand, once armed with theteaching provided herein, that the present invention encompasses amethod of preventing a wide variety of diseases or disorders whereincreased expression and/or activity of NPP4 mediates, treats orprevents a disease or disorder. Further, the invention encompassestreatment or prevention of such diseases or disorders discovered in thefuture.

The invention encompasses administration of NPP4, including an NPP4polypeptide, a recombinant NPP4 polypeptide, a mutant NPP4 polypeptide,an active NPP4 polypeptide fragment, or an NPP4 activator to practicethe methods of the invention; the skilled artisan would understand,based on the disclosure provided herein, how to formulate and administerthe appropriate NPP4 polypeptide, recombinant NPP4 polypeptide, activeNPP4 polypeptide fragment, or NPP4 activator to a subject. However, thepresent invention is not limited to any particular method ofadministration or treatment regimen. This is especially true where itwould be appreciated by one skilled in the art, equipped with thedisclosure provided herein, including the reduction to practice using anart-recognized model of pathological calcification or ossification, thatmethods of administering an NPP4 polypeptide, a recombinant NPP4polypeptide, a mutant NPP4 polypeptide, an active NPP4 polypeptidefragment, or NPP4 activator can be determined by one of skill in thepharmacological arts.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Cloning and Expression

NPP1 and NPP4 are transmembrane proteins localized to the cell surfacewith distinct intramembrane domains. For example, NPP1 is in a type-IIorientation while NPP4 is in a type-I orientation. In contrast, NPP2 issynthesized as a pre-pro-protein, and following proteolytic processingis secreted as a soluble protein following cleavage of the extracellulardomain by furin (Jansen et al., 2005, J. Cell Sci. 118:3081-3089). Toexpress NPP4 as a soluble, recombinant protein in baculovirus, thecytoplasmic and transmembrane domains were omitted from the proteinconstruct. In contrast, to express NPP1 as a soluble extracellularprotein, the transmembrane domain of NPP1 was swapped for thetransmembrane domain of NPP2, which resulted in the accumulation ofsoluble, recombinant NPP1 in the extracellular fluid of the baculoviruscultures.

NPP1 can be made soluble by omitting the transmembrane domain. HumanNPP1 (NCBI accession NP_006199) was modified to express a soluble,recombinant protein by replacing its transmembrane region (e.g.,residues 77-98) with the corresponding subdomain of human NPP2 (NCBIaccession NP_001124335, e.g., residues 12-30). The modified NPP1sequence was cloned into a modified pFastbac HT vector possessing a TEVprotease cleavage site followed by a C-terminus 9-HIS tag, and clonedand expressed in insect cells, and both proteins were expressed in abaculovirus system as described previously (Albright et al., 2012, Blood120:4432-4440; Saunders et al., 2011, J. Biol. Chem. 18:994-1004;Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362), resulting in theaccumulation of soluble, recombinant protein in the extracellular fluid(FIGS. 2-3).

Sequences NPP1 Amino Acid Sequence (NCBI accessionNP_006199) (SEQ ID NO: 1)MERDGCAGGGSRGGEGGRAPREGPAGNGRDRGRSHAAEAPGDPQAAASLLAPMDVGEEPLEKAARARTAKDPNTYKVLSLVLSVCVLTTILGCIFGLKPSCAKEVKSCKGRCFERTFGNCRCDAACVELGNCCLDYQETCIEPEHIWTCNKFRCGEKRLTRSLCACSDDCKDKGDCCINYSSVCQGEKSWVEEPCESINEPQCPAGFETPPTLLFSLDGFRAEYLHTWGGLLPVISKLKKCGTYTKNMRPVYPTKTFPNHYSIVTGLYPESHGIIDNKMYDPKMNASFSLKSKEKFNPEWYKGEPIWVTAKYQGLKSGTFFWPGSDVEINGIFPDIYKMYNGSVPFEERILAVLQWLQLPKDERPHFYTLYLEEPDSSGHSYGPVSSEVIKALQRVDGMVGMLMDGLKELNLHRCLNLILISDHGMEQGSCKKYIYLNKYLGDVKNIKVIYGPAARLRPSDVPDKYYSFNYEGIARNLSCREPNQHFKPYLKHFLPKRLHFAKSDRIEPLTFYLDPQWQLALNPSERKYCGSGFHGSDNVFSNMQALFVGYGPGFKHGIEADTFENIEVYNLMCDLLNLTPAPNNGTHGSLNHLLKNPVYTPKHPKEVHPLVQCPFTRNPRDNLGCSCNPSILPIEDFQTQFNLTVAEEKIIKHETLPYGRPRVLQKENTICLLSQHQFMSGYSQDILMPLWTSYTVDRNDSFSTEDFSNCLYQDFRIPLSPVHKCSFYKNNTKVSYGFLSPPQLNKNSSGIYSEALLTTNIVPMYQSFQVIWRYFHDTLLRKYAEERNGVNVVSGPVFDFDYDGRCDSLENLRQKRRVIRNQEILIPTHFFIVLTSCKDTSQTPLHCENLDTLAFILPHRTDNSESCVHGKHDSSWVEELLMLHRARITDVEHITGLSFYQQRKEPVSDILKLKTHLPTFSQED NPP2 Amino Acid Sequence (NCBI accessionNP_001124335) (SEQ ID NO: 2)MARRSSFQSCQIISLFTFAVGVNICLGFTAHRIKRAEGWEEGPPTVLSDSPWTNISGSCKGRCFELQEAGPPDCRCDNLCKSYTSCCHDFDELCLKTARGWECTKDRCGEVRNEENACHCSEDCLARGDCCTNYQVVCKGESHWVDDDCEEIKAAECPAGFVRPPLIIFSVDGFRASYMKKGSKVMPNIEKLRSCGTHSPYMRPVYPTKTFPNLYTLATGLYPESHGIVGNSMYDPVFDATFHLRGREKFNHRWWGGQPLWITATKQGVKAGTFFWSVVIPHERRILTILQWLTLPDHERPSVYAFYSEQPDFSGHKYGPFGPEMTNPLREIDKIVGQLMDGLKQLKLHRCVNVIFVGDHGMEDVTCDRTEFLSNYLTNVDDITLVPGTLGRIRSKFSNNAKYDPKAIIANLTCKKPDQHFKPYLKQHLPKRLHYANNRRIEDIHLLVERRWHVARKPLDVYKKPSGKCFFQGDHGFDNKVNSMQTVFVGYGSTFKYKTKVPPFENIELYNVMCDLLGLKPAPNNGTHGSLNHLLRTNTFRPTMPEEVTRPNYPGIMYLQSDFDLGCTCDDKVEPKNKLDELNKRLHTKGSTEAETRKFRGSRNENKENINGNFEPRKERHLLYGRPAVLYRTRYDILYHTDFESGYSEIFLMPLWTSYTVSKQAEVSSVPDHLTSCVRPDVRVSPSFSQNCLAYKNDKQMSYGFLFPPYLSSSPEAKYDAFLVTNMVPMYPAFKRVWNYFQRVLVKKYASERNGVNVISGPIFDYDYDGLHDTEDKIKQYVEGSSIPVPTHYYSIITSCLDFTQPADKCDGPLSVSSFILPHRPDNEESCNSSEDESKWVEELMKMHTARVRDIEHLTSLDFFRKTSRSYPEILTLKTYLHTYESEINPP4 Amino Acid Sequence (NCBI accession AAH18054.1) (SEQ ID NO: 3)MKLLVILLFSGLITGFRSDSSSSLPPKLLLVSFDGFRADYLKNYEFPHLQNFIKEGVLVEHVKNVFITKTFPNHYSIVTGLYEESHGIVANSMYDAVTKKHFSDSNDKDPFWWNEAVPIWVTNQLQENRSSAAAMWPGTDVPIHDTISSYFMNYNSSVSFEERLNNITMWLNNSNPPVTFATLYWEEPDASGHKYGPEDKENMSRVLKKIDDLIGDLVQRLKMLGLWENLNVIITSDHGMTQCSQDRLINLDSCIDHSYYTLIDLSPVAAILPKINRTEVYNKLKNCSPHMNVYLKEDIPNRFYYQHNDRIQPIILVADEGWTIVLNESSQKLGDHGYDNSLPSMHPFLAAHGPAFHKGYKHSTINIVDIYPMMCHILGLKPHPNNGTFGHTKCLLVDQWCINLPEAIAIVIGSLLVLTMLTCLIIMVIQNRLSVPRPFSRLQLQEDDDD PLIG

Purification

The protein was purified by nickel affinity column, and followingelution with imidazole, the C-terminal histidine tag was cut from theprotein with tobacco etch virus (TEV) protease. Following cleavage ofthe histidine tag, a second round of purification on a nickel column wasperformed to remove the C-terminal histidine tag and contaminatingproteins which non-specifically associate with the nickel column duringthe first round of purification. Soluble NPP1 elutes in the flow throughand is collected and concentrated by spin concentration. The overallpurification yields approximately 2 mg of pure protein per liter of cellculture (FIGS. 3-4). A similar general purification scheme has beendescribed for biochemical, biophysical, and physiologic studies ofseveral members of the NPP family (Albright et al., 2012, Blood120:4432-4440; Saunders et al., 2011, J. Biol. Chem. 18:994-1004;Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362). Purification ofthe proteins containing the Fc domain of IgG may also be accomplishedvia binding to protein A or protein G columns, as known to thoseexperienced in the art and science of protein purification.

Mammalian Expression System for NPP1

NPP1 is a glycosylated protein, and insect cell sugar moieties areexpected to induce strong immunogenic reactions from a mammalian host.To reduce the possibility of inducing immune reactions by treatinganimals with recombinant NPP1, a mammalian expression system was used toreplace insect cell glycosylation patterns with mammalian glycosylationpatterns. The protein in the HEK293 mammalian kidney cell line wasexpressed by cloning the identical NPP1 construct into a mammalianexpression vector followed by stable transfection into HEK293 cells.Stable clones were identified by immunoblot against His antibody.Strongly expressing clones were expanded and culture medium wascollected and treated as described in the baculovirus purificationscheme described elsewhere herein. The overall yield of protein wasapproximately 1.5 mg/liter of culture media, and the purity of thesample was greater than 95% (FIG. 4).

ATP Hydrolytic Activity of NPP1 and NPP4

To verify that the extracellular, soluble domain of NPP1 isenzymatically active, the steady-state Michaelis-Menten enzymaticconstants of NPP1 were determined using ATP as a substrate. In addition,to illustrate substrate specificity between NPP family members, the ATPhydrolysis of NPP1 was directly compared with NPP4, a protein with 38%sequence identity to NPP1. To verify that NPP1 cleaved ATP, HPLCanalysis of the enzymatic reaction was used, and the identity of thesubstrates and products of the reaction was confirmed by using ATP, AMP,and ADP standards (FIGS. 5A-5E). The ATP substrate degrades over time inthe presence of NPP1, with the accumulation of the enzymatic product AMP(FIGS. 5A-5E). Using varying concentrations of ATP substrate, theinitial rate velocities for NPP1 were derived in the presence of ATP,and the data was fit to a curve to derive the enzymatic rate constants(FIG. 5C). In spite of significant sequence identity between NPP4 andNPP1, NPP4 had no ATP hydrolytic activity, while NPP1 readily hydrolyzedATP into AMP and PPi (FIG. 5C). At physiologic pH, the kinetic rateconstants of NPP1 are Km=144 μM and k_(cat)=7.8 s⁻¹.

HPLC Protocol

The HPLC protocol used to measure ATP cleavage by NPP1 and NPP4, and forproduct identification, was modified from the literature (Stocchi etal., 1985, Anal. Biochem. 146:118-124). The reactions containing varyingconcentrations of ATP in 50 mM Tris pH 8.0, 140 mM NaCl, 5 mM KCl, 1 mMMgCl₂ and 1 mM CaCl₂ buffer were started by addition of 0.2-1 μM NPP1and quenched at various time points by equal volume of 3M formic acid,or 0.5N KOH and re-acidified by glacial acetic acid to pH 6. Thequenched reaction solution was diluted systematically, loaded onto aHPLC system (Waters, Milford Mass.), and substrates and products weremonitored by UV absorbance at 254 or 259 nm. Substrates and productswere separated on a C18, 5 μm 250×4.6 mm HPLC column (HigginsAnalytical, Mountain View, Calif.), using 15 mM ammonium acetate pH 6.0solution, with a 0% to 10% (or 20%) methanol gradient. The products andsubstrate were quantified according to the integration of theircorrespondent peaks and the formula:

$\left\lbrack {{product}\text{/}{substrate}} \right\rbrack = {\frac{{area}_{{product}/{substrate}}/ɛ_{{product}/{substrate}}}{{area},{\ldots \;/ɛ},{\ldots + {area}},{\ldots \;/ɛ},\ldots}\left\lbrack {substrate} \right.}$

where [substrate]₀ is the initial substrate concentration. Theextinction coefficients of AMP, ADP and ATP used in the formula were15.4 mM⁻¹ cm⁻¹. If monitoring at 254 nm, substrate and product standardsrun on the same day as the reactions were used to convert integratedproduct/substrate peak areas to concentrations.

Mouse Models

TTW mice were discovered in brother-sister mating of Institute of CancerResearch strain mice (ICR, Japan), and develop multiple progressiveabnormal calcifications and finally succumb to severe deformation andankylosis. These mice serve as an established animal model of both OPLLand osteoarthritis because of specific spinal and joint abnormalities.The genetic defect accounting for the ectopic tissue mineralizationphenotype has been traced to a premature stop codon in NPP1 at position568 (glycine to stop), which truncates NPP1 by approximately 350 aminoacids. A variant of the TTW mouse—C57BL/6J-Enpp1^(asj)/Grsr, orNPP1^(asj) mice—has a point mutation in the NPP1 protein coding regionat exon 7, position 737, which results in a valine to an alanine pointmutation, and exhibit arthritis of the spine at 12 weeks, andosteoarthritis of many joints that become stiff and unbendable by 7months of age. These mouse models are valuable reagents which mimichuman diseases of ectopic calcification, including osteoarthritis, andare appropriate animal models to validate a hypothesis of the presentstudy.

The therapeutic efficacy of recombinant NPP1 in two mouse models ofectopic calcification, both of which are variants of tip toe walking(ttw) mice, is explored. The first model is the original ttw mouse modeldescribed by Japanese researchers, which is available through theCentral Institute for Experimental Animals in Japan. The mice have aspontaneous recessive mutation which predisposes the animals to ageneralized ankylosis of the axial and appendicular skeleton starting atthree weeks of age in the small distal joints of the hands and feet.Besides articular and vertebral disk cartilage, ttw mice also showcalcification of vessels and connective tissues. After genetic mapping,the mutation responsible for the phenotype was identified as atruncation of NPP1 at position 568. The second mouse model, directlyavailable from Jackson Laboratories, is named theC57BL/6J-Enpp1^(asj)/GrsJ mouse. These mice have a point mutation in theNPP1 protein coding region at exon 7, position 737, which results in avaline to an alanine point mutation, and exhibit arthritis of the spineat 12 weeks, and osteoarthritis of many joints which become stiff andunbendable by 7 months of age.

Establishment of NPP1 dosing levels

NPP1 intra-peritoneal (IP) dosing levels that normalize serum PPiconcentrations in ttw mice are established. Initial pharmacokineticexperiments are performed to establish the appropriate route andconcentration of NPP1 necessary to affect PPi levels in vivo. Wild type(C57bl/6) animals are dosed with a single dose of NPP1 (e.g., i.p.,i.v., etc.) at concentrations beginning at 0.03 mg/kg. This initialconcentration is chosen because physiologic NPP1 concentrations in humanserum are quite low (between 100-300 ρM). A concentration of mammalianNPP1 of 2.15 mg/ml in an aqueous buffer is in close proximity tophysiologic conditions (50 mM Tris pH 8.0, 150 mM NaCl, 0.8 mM ZnCl₂,0.4 mM CaCl₂, 0.4 mM MgCl₂). For instance, a 40 gram mouse would beinjected with about 0.5 μl of concentrated stock. NPP1 dosing isadjusted as necessary to arrive at dosing levels that directly affectserum PPi concentrations.

About three animals are dosed per experimental group. The animals aredosed at about 0, 0.5 mg/kg, 2 mg/kg, and 8.0 mg/kg concentrations tobegin. Animals are sacrificed at about 1, 4, and 8 hours post treatment,and blood is collected by cardiac puncture following terminalanesthesia. Serum is isolated, and serum concentrations of PPi aredirectly measured using a commercially available fluorogenicpyrophosphate sensor that has its fluorescence intensity proportionallydependent upon the concentration of pyrophosphate (see Abcam productnumber ab112155). Serum pyrophosphate concentrations of dosed andundosed animals are compared to determine initial dosing levels of NPP1that physiologically alter serum PPi values in mice.

Pharmacokinetic experiments with the ttw mice are undertaken once NPP1dosing levels are established in the wild type mice. To start, NPP1concentrations which modulate PPi levels in wild type mice are used.About three animals are used per experimental group, and the animals aredosed at about 0, 1X, and 10X levels, where X is the minimal NPP1concentration observed to modulate serum PPi levels in wild type(c57bl/6) mice. Animals are sacrificed at about 1, 4, and 8 hours posttreatment, blood is collected and PPi concentrations are analyzed asdescribed elsewhere herein. The serum PPi levels of treated anduntreated animals are compared with those of the wild type animals inexperiments described elsewhere herein to establish the final beginningconcentrations of NPP1 to be used in the efficacy experiments.

Efficacy of Recombinant NPP1 in Mouse Models of Ectopic Calcification

TTW and C57BL/6J-Enpp1^(asj)/GrsJ mice are used to determine the effectof recombinant NPP1 on mouse models of ectopic calcification, asdescribed elsewhere herein. Four breeding pairs are established todevelop each genetic colony. Because the onset of progressive physicaldisability impairs the ability of homozygous females to maintain alitter, heterozygous females are bred with homozygous males. The animalsare genotyped by Yale Animal Resource Center at three weeks of age, andonce weaned the animals are separated into cages according to theirgenotypes and ages. Strict record keeping is maintained to ensureanimals are correctly identified prior to experimentation. At 6 weeks ofage the mice are separated into cohorts of about 6 mice and are treatedwith increasing concentrations of NPP1, beginning at the lowestconcentrations observed to normalize serum PPi concentrations, asdescribed elsewhere herein.

The development of symptoms is compared between treated and untreatedanimals, with attention paid to the following phenotypes:

Gait: A slow, hobbling gait in the C57BL/6J-Enpp1^(asj)/GrsJ mice occursat around 2 months of age. TTW mice also develop abnormal gait, rigidityof the vertebral column, and stiffness of the limb joints at about 2months of age.

Abnormal Resting Posture: C57BL/6J-Enpp1^(asj)/GrsJ mice develop a stiffposture with the front legs held in toward the body by about 2 months ofage. TTW mice also develop stiffness of the limb joints at about 2months.

Skeleton Phenotype: C57BL/6J-Enpp1^(asj)/GrsJ mice develop hyperplasticjoint spaces in the knees and elbows at about 11 weeks, arthritis of thespine at about 12 weeks, and osteoarthritis in many joints at about 12weeks. By about 7 months, the joints of C57BL/6J-Enpp1^(asj)/GrsJ micebecome stiff and unbendable. TTW mice develop rigidity of the vertebralcolumn, and generalizing ankylosis of the axial and appendicularskeleton starting at about 3 weeks of age.

Osteoartheritis: Osteoartheritis is found in both ttw andC57BL/6J-Enpp1^(asj)/GrsJ mice at approximately 12 weeks of age.

Hearing Loss: Auditory brainstem response shows severe hearing loss inC57BL/6J-Enpp1^(asj)/GrsJ mice by about 3 months of age.

Imaging studies: Sites of active mineralization in mice and man may beobserved with radionucleotide scans utilizing various agents, includingby not limited to Tc99m-pyrophosphate. Increased Tc99m-pyrophosphate maybe seen in animals with increased mineralization. Radionucleotideimaging scans are performed on the animals at day 0, 7, and 14, oftreatment by subclavian injection of the radiolabelled tracer. Increasesin PPi deposition are correlated with NPP1 dosing levels. In certainembodiments, PPi deposition increases in NPP1 mutant animals at thelowest concentration levels of recombinant NPP1 or NPP4 enzyme, buttherapeutic levels of recombinant NPP1 or NPP4 reduce or eliminate PPideposition as observed in radionucleotide scans.

At least one of the above phenotypes are monitored and/or quantified inthe treated and untreated animals by recording the gait of animals ontreadmills to follow gait and posture, imaging studies to follow in vivoskeletal changes during the course of the experiment, auditoryassessment of the mice to document hearing loss, and histologicexamination of the skeleton and soft tissues of the mice at theconclusion of the experiment to document ectopic calcification andskeletal abnormalities. MRI imaging is used to follow the extent of softtissue mineralization and skeletal abnormalities in the animals duringthe course of the experiment. Prior to imaging, the mice areanesthetized with isofluorane in an anesthesia chamber, and thentransferred to an IVIS imager box during the imaging process. The miceare maintained under anesthesia via nose cones that are present in theMRI imager box during the imaging process. The animals are observed forabout 30 minutes post procedure for signs of pain and distress followingreversal of anesthesia. Mice require 1-2 minutes to recover from theanesthesia, and that the mice can be safely imaged 2-3 times per weekfor up to 1 month without health sequella. Mice are periodicallyrecorded on mechanized treadmills in order to observe and analyze theirgait and the presence of postural changes. Auditory brain response andthe lick suppression test are used to monitor hearing loss in theanimals, and tissue and bone histology is used to document the presenceof soft tissue calcification and bone abnormalities.

As described elsewhere herein, steps are taken to minimize immunereaction to the recombinant human NPP1 in the immunocompetent ttw mice.In the event that unexpected decreases in serum PPi concentrations areobserved, which are consistent with a reduced NPP1 half-life suggestiveof immune mediated destruction of the protein, standard approaches ofimmune-tolerization can be used, such as those which have been used inmice to generate monoclonal antibodies specific to alternate proteinisoforms (Matthew et al., 2987, J. Immuno. Methods 100:73-82; Salata etal., 1992, Anal. Biochem. 207:142-149). These procedures involveimmunizing the animals with cyclophosphamide concurrent with the initialdose of NPP1 protein, which can tolerize the animal to the humanisoform. If tolerization is unsuccessful, the mouse isoform of NPP1 iscloned and expressed in an identical manner to that described for thehuman, and the mouse isoform of the protein is used to conduct thedescribed experiments. Either of these alternative procedures provides amethod of treating mice with NPP1 in a manner that will not induce animmunologic response, thereby achieving stable, efficacious NPP1concentrations sufficient to delay or reverse the ectopic mineralizationcharacterizing their genetic disease.

Crystallization, Data Collection and Processing, Structure Determination

NPP4 was exchanged into 50 mM Tris pH 8.0, 150 mM NaCl, 0.8 mM ZnCl₂,0.4 mM CaCl₂, 0.4 mM MgCl₂), and protein concentrations were calculatedusing A₂₈₀. The best diffracting crystals were obtained via hanging dropmethod by mixing 6 mg/ml (0.14 mM) of NPP4 with well solution (200 mMammonium citrate dibasic, 17.5% to 19.5% (w/v) PEG 3350) in a 1:1 ratioand suspending 2 μl drops over 600 μl of the well solution in a sealedchamber. Protein crystals typically appeared within 4 to 6 days andcontinued to grow slowly for the next week, reaching final dimensions ofup to 500 um×150 um×50 um. Cryoprotection was achieved by quicklypassing the crystals through a series of mixtures consisting of theabove well solution with 0.6 mM ZnCl₂, 5 mM ligand (if present), and 5%(v/v) increments of glycerol up to a final concentration of 20% to 25%,then immediately flash-freezing in liquid nitrogen. Apo crystals of NPP4were difficult to obtain, hampering efforts to obtain structures of NPP4with ligands via soaking. Cocrystallization with ATP or a cleavableATP-analogue (Sigma M7510) resulted in an AMP product complex,reflecting slow hydrolysis during crystallization. Cocrystallizationattempts with a non-cleavable ATP-analogue (Sigma M6517) showed novisible binding.

Synchrotron diffraction data reported herein were collected at APS(Argonne National Laboratory, Advanced Photon Source, NE-CAT beamlinesID-24-C and ID-24-E), and at CHESS (Cornell High Energy SynchrotronSource, beamline A1). HKL2000 was used for indexing and reduction of theNPP4-AMP diffraction data. Initial phases were obtained via molecularreplacement using PHENIX AutoMR and AutoBuild with a search modelconsisting of the protein-only portion of the Xanthomonas axonopodis NPP(2GSU) (Zalatan et al., 2006, Biochem. 45:9788-9803). COOT was employedfor model building and the high quality of initial electron density mapsallowed for unambiguous correction of areas of the structure that wereoriginally problematic or out of sequence. PHENIX was used for iterativerounds of maximum likelihood refinement during which ligand, waters andseveral glycosylations were built in. Apo NPP4 and all subsequentstructures were solved as above, but using the protein-only portion ofthe NPP4-AMP complex as the starting point. Unbiased electron densitymaps in which the local atoms were excluded from map calculations (omitmaps) were used to systematically check each entire structure. Atomicpositions for all residues 24 to 402 were determined. Flanking residuesat the termini remain disordered. Statistics for diffraction data thefinal structures are illustrated in Table 1. The structures of humanNPP4-AMP at 1.54 Å resolution (4LQY) and apo NPP4 at 1.50 Å resolution(4LR2) have been deposited in the Protein Data Bank.

Molecular Modeling of NPP1:ATP Complex

Mouse NPP1 (rcsb code 4B56) was loaded in MOE (Molecular OperatingEnvironment, Chemical Computing Group Inc., Montreal, Canada) and theA-chain deleted and B chain retained with residues K169-E905.Asparagine-linked glycosylation sites were clipped and capped withmethyl groups. Waters were deleted and atom types fixed and protonatedwith Protonate 3D. Zinc ions were restrained with respect to theirligand distance and geometry. The metal charge was modeled at +1. ATPwas manually positioned by placing the adenine nucleus between the cleftformed from phenylalanine 239 and tyrosine 322 with the phosphateportion in an extended conformation along the large channel moving upthe protein. The protein was tethered at a distance of 4.5 angstromsfrom the ligand and minimization performed on the entire system usingAMBER12 with Extended Huckel Treatment of the ligand (AMBER12:EHT).

Molecular modeling of NPP4:ATP complex

NPP4 was modeled in a manner similar to NPP1 including glycosylstripping and capping and placement of ATP. The corresponding residuesin NPP4 for placement of the adenine are phenylalanine 71 and tyrosine154. Protein protonation, tethering and minimization was performed in ananalogous fashion, using the AMBER12:EHT forcefield treatment.

Enzymology

The steady state enzymatic activity of human NPP1 and NPP4 weredetermined by either absorbance (Ap3A substrate) or HPLC (ATPsubstrate). The affinities of nucleotide monophosphates (NMP) for NPP4were estimated from the [NMP] dependence of steady state pNP-TMP(p-nitrophenyl 5′ thymidine monophosphate) cleavage rates as monitoredby the absorbance change at 405 nm (Saunders et al., 2008, Mol. CancerTher. 7:3352-3362). The [NPP4] was 5 nM and the [pNP-TMP] was 20 mM. TheIC₅₀ value (i.e. nucleotide concentration exhibiting half maximalactivity) was determined from the best fit of the nucleotideconcentration dependent NPP4 cleavage activity to a rectangularhyperbola. The IC₅₀ reflects the weighted average affinity for mixedinhibition (Saunders et al., 2008, Mol. Cancer Ther. 7:3352-3362).

Preparation of Human Platelets and Platelet Aggregometry

Preparation of platelets and platelet aggregometry were performed asdescribed in Albright et al., 2012, Blood 120:4432-4440.

Example 1: Enzyme Replacement Therapy for Idiopathic Infantile ArterialCalcification (IIAC) and Ossification of the Posterior LongitudinalLigament (OPLL)

As described herein, a soluble form of the nucleotidepyrophosphatase/phosphodiesterase-1 (NPP-1) and mutant NPP-4 has beendeveloped, which can be a useful therapeutic for diseases and disordersinvolving pathological calcification and/or ossification. Also describedherein is a direct demonstration of the physiologic activity of theenzyme in disease states of NPP1 dysfunction, thereby establishing theutility of NPP1 enzyme replacement therapy in select disorders ofectopic calcification.

To develop a soluble form of NPP1, the sequences of NPP2 were combinedwith the sequences of NPP1 to obtain a soluble, secreted NPP1 protein.It was discovered that soluble, active, recombinant NPP1 could beproduced by swapping the membrane-spanning domain of NPP2 into thehomologous region of NPP1. Using such a construct, some variant forms ofNPP1 occurring in OPLL and IIAC have been cloned and expressed. In someembodiments, NPP1 can be made to be soluble by removing the entiretransmembrane domain of the protein.

Soluble, fully active forms of NPP1, and mutant NPP4 engineered tochange the specificity of NPP4 from Ap3A to ATP, are useful proteintherapeutics in OPLL, IIAC, and other diseases resulting from improperPPi balance. Specifically, the protein construct is comprised of thesoluble domain of NPP1, the constant region of human IgG Fc domain (Fc),and about ten or more sequential Aspartic acid residues designed totarget the protein to the bone.

These constructs yield soluble forms of NPP1 and mutant NPP4 at highyields, resulting in large quantities of soluble, pure, recombinant,enzymatically active NPP1 and mutant NPP4 capable of hydrolyzing ATPinto AMP and PPi. These constructs may be useful in treating humandiseases and disorders with improper PPi balance which are alsoassociated with SNPs in NPP1.

The structure of NPP1 was modeled using the high resolution structure ofNPP4 which had been previously determined, and it was discovered thatmany of the SNPs associated with the diseases described elsewhere hereinmap to the active site of NPP1.

Although not wishing to be bound by any particular theory, the datadescribed herein are consistent with the explanation that reduced NPP1activity is responsible for disrupting PPi/Pi homeostasis in many of thedisorders described elsewhere herein, and that restoring PPi balancethrough the use of soluble, recombinant, NPP1 protein ameliorates theskeletal and joint abnormalities and ectopic tissue mineralization seenin these diseases.

OPLL is examined using standard animal models of the human disease withso called ‘tip-toe walking’ (ttw) mice. These mice develop progressiveabnormal calcifications of the spine and joints that bear a strikingresemblance to OPLL disease in humans, and eventually succumb to severespinal deformations and ankylosis. The genetic defect of thisspontaneously occurring mutation introduces a missense mutation into thecoding region of NPP1 at position 568, prematurely truncating theprotein. These mice are the accepted animal model for OPLL.

Dosing routes and concentrations of recombinant NPP1 in these mice whichnormalize their serum PPi concentrations are established, and whetherthese doses are able to defer, ameliorate, or reverse the signs and/orsymptoms of the severe phenotype experienced by these animals is tested.NPP1 is examined as a protein therapeutic for animal models of ectopictissue mineralization, including OPLL and osteoarthritis. The efficacyof recombinant NPP1 in diseases of ectopic tissue calcification isestablished by measuring the rate of joint and spine calcification intip-toe walking (ttw) mice treated with recombinant NPP1 protein.

The appropriate NPP1 dosing necessary to normalize PPi levels isestablished by dosing ttw and wild type (c57bl/6, the geneticbackground) mice with increasing NPP1 concentrations and measuring theserum PPi concentrations in treated and untreated animals usingestablished methods of PPi determination. The efficacy of recombinantNPP1 in diseases of ectopic calcification is determined by treating ttwmice with recombinant NPP1 and comparing the development of symptomsassociated with ectopic tissue calcification between the treated anduntreated animals.

Example 2: Substrate Discrimination of NPP4 Vs. NPP1

To determine the extent of substrate discrimination exhibited by highlyhomologous NPP family members, the steady state enzymatic rate of humanNPP4 and NPP1 for their putative in vivo substrates, Ap3A and ATP,respectively, was measured (FIGS. 5A-5E). Human NPP4 shares 40% sequenceidentity with NPP1 throughout the catalytic domain, and the structure ofmouse NPP1 has been determined (Jansen et al., 2012, Structure20:1948-1959), providing the opportunity to identify the structuralorigins of substrate discrimination at the atomic level. Human and mouseNPP1 are 79% identical, with sequence mapping of the human sequence ontothe mouse NPP1 structure showing that all sequence differences areoutside the substrate binding and active sites. NPP4 and NPP1 hydrolyzeAp3A with comparable maximum turnover numbers (k_(cat)˜7-8 s⁻¹),although the Michaelis constant of NPP1 is >30 times tighter than thatof NPP4. In contrast, the rate at which NPP4 hydrolyzes ATP to AMP andPPi is negligible compared to hydrolysis by NPP1.

Example 3: Structural Overview

In order to understand the molecular basis of NPP4 substrate specificityand to achieve detailed insights regarding its similarities anddifferences relative to NPP1, X-ray crystallography was utilized todetermine the high-resolution three-dimensional structures of human NPP4in both apo and AMP-bound forms, at 1.50 Å and 1.54 Å resolution,respectively (Table 1 and FIG. 6A). These were then compared to recentlydetermined structures of mouse NPP1 complexes, including mNPP1-AMP at2.70 Å resolution, in order to define structural features responsiblefor the observed substrate specificities of each. The bimetallocatalytic domains of NPP4 and NPP1 contain two bound zinc ions, andshare a similar overall fold and employ a conserved catalytic mechanismto hydrolyze at the same position on substrates, resulting in anucleotide monophosphate product. Hydrolysis of Ap3A or ATP by either ofthese enzymes yields an AMP product molecule. Accordingly, hNPP4-AMP andmNPP1-AMP structures are product-complexes. Superposition of the entirecatalytic domain of NPP4 with that of NPP1, NPP2 and the bacterial NPPyield rmsd values of 1.54 Å, 1.43 Å and 1.43 Å, respectively. Thestructural features observed in NPP4 that favor nucleotide binding arelargely conserved in the bacterial enzyme.

NPP4 is a monomeric enzyme with a binding pocket that remainsessentially unchanged in the presence or absence of bound product.Disulfide bonds link residues 254 to 287 and 394 to 401, and threeN-linked glycosylations are observed at asparagine residues 155, 166 and386. Due to location, these glycosylations are not likely tosignificantly impact enzymatic activity. This is the first human NPPstructure to be determined and shares an overall common fold with thecatalytic domains of all other structurally determined NPPs (FIG. 6B).

Example 4: Substrate Recognition and Active Site Geometry

NPP4 targets phosphodiester substrates with a 5′-nucleotide group on theend, displaying the greatest preference for adenine rings. Thisspecificity for nucleotide-containing substrates is the result of apre-formed hydrophobic slot on the protein surface, approximately 6.8 Åwide, consisting of the ring face of Tyr154 on one side and the tip ofPhe71 on the other (FIGS. 7A-7B). A nucleotide base bound withinexperiences favorable π-π stacking interactions with the tyrosine ringand van der Waals (VDW) interactions with the phenylalanine, sittingabout 3.4 Å from each. The back wall of the slot lies just beyond thereach of the nucleotide base, precluding any direct hydrogen bondinginteractions with the protein, and only a couple of water-mediatedhydrogen bonds are observed between the edge of the AMP ring and NPP4.

Although such a slot might be expected to favor purines over pyrimidinesby virtue of their greater size, this pattern does not strictly hold.The relative affinities of nucleotide monophosphates for NPP4, asdetermined by TMP-pNP substrate inhibition, are: AMP>CMP>UMP>GMP (FIG.5E). Corresponding measurements reported for NPP1, another family memberwith a similar nucleotide slot, reveal: AMP>CMP>GMP>UMP (Kato et al.,2012, Proc. Natl. Acad. Sci. USA 109:16876-16881). The present modelbuilding reveals that the guanine ring N2 atom of a similarly-bound GMPwould experience some steric clash with NPP4 at residues 104 and 105,which slightly overhang the nucleotide slot. Upon inspection of theNPP1-GMP cocrystal structure, a similar steric clash was discovered,forcing the guanine ring to rotate slightly.

Immediately adjacent to the hydrophobic slot are two bound zinc ions,hereafter referred to as Zn1 and Zn2, held ˜4.5 Å apart via interactionswith six invariant residues found in all NPPs and alkaline phosphatases(APs). Both zinc ions display tetrahedral coordinate geometry. Zn1 isligated by Asp189, His193 and His336, with a fourth coordination comingfrom a phosphate group oxygen atom in the NPP4-AMP complex.Alternatively, in an empty pocket this coordination can be provided by awater molecule. Zn2 is held by Asp34, Asp237 and His238, with a fourthcoordination to the Oγ atom of Thr70, the “catalytic residue” of NPP4.This close association serves to activate Thr70 for nucleophilic attackon a substrate molecule, as described for APs, presumably by perturbingits pKa. Thr70 is located at the N-terminus of an α-helix pointingdirectly at the phosphate binding site near the semi-exposed Zn1, suchthat helix-dipole forces compliment the positively-charged zinc ions inelectrostatically drawing the negatively-charged phosphodiester groupinto the active site. The narrow nucleotide slot and the short spacingbetween the slot and the zinc ions are primarily responsible forsubstrate selection, sterically favoring a 5′-nucleotide monophosphategroup on the end.

By comparison, the other end of the NPP4 binding pocket appearsrelatively featureless and significantly more solvent-exposed,consistent with the ability of NPP4 to hydrolyze substrates of varyinglength and chemical character. No crystal structures of any NPP withintact substrate bound have been reported to date, however our substratedocking simulations indicate that the NPP4 binding pocket runs along theshallow groove on the protein surface.

Example 5: NPP4-AMP Vs. Apo NPP4

The apo structure of NPP4 is essentially identical to the AMP-boundstructure (FIG. 7C). Since no changes are observed within the emptyhydrophobic slot or at the catalytic residue, the binding site ispre-formed and appears to not undergo induced-fit adjustments uponsubstrate binding. The apo structure has a citrate anion, from thecrystallization conditions, bound at Zn1 in a chelation-likeinteraction. Asn91 is the only active site residue that moves notably,pivoting to allow room for the citrate molecule. In the NPP4-AMPcomplex, Asn91 donates a hydrogen bond to the phosphate group bound nearZn1. The ability of Asn91 to pivot may allow it to maintain a hydrogenbonding interaction with the phosphate oxygen through the catalyticintermediates of the reaction, or allow flexibility to facilitate theentry or exit of a ligand.

Example 6: NPP4-AMP Vs. NPP1-AMP

The overall similarity of how AMP binds within the slot region of NPP4and NPP1 is illustrated in FIG. 7D. NPP1 contains conserved residuescorresponding to Tyr154 and Phe71 of NPP4, therefore also targetssubstrates with a 5′-nucleotide end. The phosphate group of AMP bindsnear Zn1 in both enzymes. Whether the small differences observed in AMPposition are real or simply reflect the significantly lower resolutionof the NPP1-AMP structure (2.70 Å versus 1.54 Å for NPP4-AMP) isunknown.

Example 7: Catalytic Mechanism

NPPs are members of the AP superfamily and share many of the keystructural features, residues and architecture at the center of theactive site where the catalysis occurs. FIG. 8 depicts a generalreaction mechanism for NPPs as it applies to NPP4 hydrolysis of Ap3A,along with the corresponding crystal structures or binding simulationmodel for each step.

In NPP4, the relative spacing between the pre-formed hydrophobic slotand the two bound zinc ions dictates that it is the α-phosphate group ofthe substrate that is positioned adjacent to the Zn1 and Thr70, whose Oγatom is perpetually activated for nucleophilic attack by its closeproximity to Zn2. Upon Ap3A binding, the α-phosphate is attacked byThr70, causing the ester bond on the opposite side to break, releasingADP. A water molecule immediately enters the vacant space next to Zn1,becomes activated and attacks the α-phosphate from the oppositedirection, causing the transient covalent NPP4-AMP bond to break,releasing AMP and restoring NPP4 to its original state. The release ofthe two product molecules is sequential, with the nucleotidemonophosphate leaving last. As such, NPP4-AMP and NPP1-AMP areproduct-complexes.

Example 8: Molecular Determinants of Substrate Specificity of NPP4 andNPP1

NPP1 readily cleaves ATP into AMP and PPi, whereas NPP4 does so onlyexceedingly slowly. Since both enzymes target nucleotide-containingsubstrates with a preference for adenine rings, and bind AMP in asimilar fashion (FIG. 7D), the key to this deviation must lie elsewherewithin the binding pocket. Since NPP1 efficiently hydrolyzes ATP intoAMP and PPi, ATP likely binds NPP1 in the same orientation as isobserved for AMP. ATP was thus modeled into the NPP1 active site byadding two phosphate groups to the NPP1-AMP cocrystal coordinates (4B56)(FIGS. 9A, 9C, 9E), then subjected the complex to energy minimization asdescribed. Superposition of the binding sites of NPP1 with NPP4 revealsgood overlap between most side chains within 4.5 Å of this ATPsubstrate, but with notable differences occurring near the terminalγ-phosphate. In NPP1, donation of Phe516 (mouse numbering) to theprotein core creates more space for the γ-phosphate of ATP, whichappears to be charge-stabilized by three lysine residues (Lys237, Lys260and Lys510), which are referred to as a lysine claw. Two of theselysines (Lys260 and Lys510) line the upper edge of the binding pocketand remain disordered in the absence of a γ-phosphate, as is observed inNPP1-AMP structure. Ther simulation illustrates that, as new substrateenters the NPP1 binding site, these lysines should be electrostaticallydrawn to the γ-phosphate and become ordered in the process. Theγ-phosphate of bound ATP appears to lie under an induced-fit lidcomprised of Tyr433, Lys260 and Lys510, where it should becharge-stabilized by three lysines (FIG. 9A). Upon PPi product release,Lys260 and Lys510 should again become disordered until new substrate isdrawn to the site.

In a likewise manner, ATP was modeled into the NPP4 active site byadding two phosphates onto the NPP4-AMP structure, after which thecomplex was energy minimized. In contrast to NPP1, the region of NPP4near the γ-phosphate is much more open and solvent-exposed, containsadditional negatively-charged residues and fewer positively-chargedones, and has no ability to form lysine claw or a lid (FIGS. 9B, 9D and9F). The γ-phosphate of ATP bound in this orientation would sit next tothe negatively-charged Asp335, which corresponds to Phe516 of NPP1, butnow points directly into the binding pocket. Asp264 (corresponding toVal 432, mouse NPP1) is also nearby. Overall, the local electrostaticenvironment of this region of the NPP4 binding pocket is significantlyless favorable than in NPP1.

The ability to charge stabilize at this position may be more pronouncedwith ATP than with other substrates, such as Ap3A, since a terminalphosphate group (phosphomonoester) intrinsically carries morenegative-charge than a non-terminal phosphate group (phosphodiester). Assuch, the terminal γ-phosphate of ATP carries more negative-charge thanthe corresponding in-line γ-phosphate of Ap3A. The highly effectivecharge-stabilization of NPP1 at this position is likely key to itsability to readily hydrolyze ATP, whereas the less-favorable localenvironment found at this same position in NPP4 is detrimental andhydrolysis occurs only very slowly.

Example 9: NPP1 and Platelet Aggregation

The hydrolysis of Ap3A by NPP1 raises the question of whether NPP1 mayplay a role in platelet aggregation under physiologic conditions. Giventhat the enzyme is active against Ap3A substrate, the relative abundanceof NPP1 within the vascular space may determine the role of NPP1 incoagulation. NPP1 is reported to be present on brain capillaryendothelium, but not on capillaries elsewhere. In addition, NPP1 ispresent on the membrane surfaces of plasma cells, osteoblasts,chondrocytes, and matrix vesicles (MVs) shed from osteoblasts andchondrocytes. NPP1 is also present as a soluble protein within thevasculature at very low concentrations of between 10-30 ng/mL (or100-300 pM).

To determine the concentration of NPP1 capable of inducing plateletaggregation, increasing concentrations of NPP1 were titrated intoplatelet rich plasma (PRP) with physiologic levels of Ap3A. Addition ofNPP1 to 80 uM Ap3A results in no platelet aggregation until theconcentrations of NPP1 reach 1 nM (FIGS. 10A-10B). While thisconcentration is approximately 3-fold greater than the highest reportedplasma concentrations of NPP1, this concentration is below the thresholdrequired for NPP4 to trigger platelet aggregation (FIGS. 10A-10B), andwell within the concentration range of membrane bound endothelialproteins. Plasma concentrations of NPP1 are thus unlikely to induceprimary, platelet-mediated hemostasis in vivo, but that epithelial-boundNPP1 at anatomic locations where local concentrations exceed 1 nM arelikely to induce significant platelet aggregation and thrombusformation.

Example 10: Ap3A Bound to NPP4

Human NPP4 (rcsb code 4LR2) was loaded in MOE (Molecular OperatingEnvironment, Chemical Computing Group Inc., Montreal, Canada), andasparagine-linked glycosylation sites were clipped and capped withmethyl groups. Waters were deleted and atom types fixed and protonatedwith Protonate 3D. Zinc ions were restrained with respect to theirligand distance and geometry. The metal charge was modeled at +1. Ap3Awas manually positioned by placing the adenine nucleus between the cleftformed from phenylalanine 239 and tyrosine 322 with the phosphateportion in an extended conformation along the large channel moving upthe protein. The remainder of the Ap3A molecule was left free insolution. The protein was tethered at a distance of 4.5 Å from theligand and minimization performed on the entire system using AMBER12with Extended Huckel Treatment of the ligand (AMBER12:EHT). The dockedAp3A molecule (FIG. 11) revealed the location of the second adeninebinding site. This binding site was formed by a narrow groove in theprotein surface, which in certain embodiments may be eliminated by thealteration of residues lining the surface of the pocket.

Example 11: Crystals of Ap3A Bound to NPP4

To directly determine the location of Ap3A in the NPP4 protein, aninactive mutant of NPP4 was crystallized with Ap3A (FIG. 12). Thisstructure may be used to refine mutations of NPP4 and NPP1 designed toreduce the pro-thrombotic effects of the enzymes.

TABLE 1 Data collection and refinement statistics. NPP4-AMP NPP4 apospace group C2 C2 a, b, c (Å) 181.5, 51.1, 52.7 181.5, 51.3, 53.1 α, β,γ (°) 90, 102.4, 90 90, 102.1, 90 resolution (Å) 50-1.54 50-1.50beamline APS/NE-CAT-C CHESS/A1 wavelength (Å) 1.0750 0.9779 Rsym(%)^(a, b) 6.0 (53.6) 8.4 (54.8) I/σI ^(a, c) 724.9/35.6 (37.0/13.5)503.2/28.0 (19.0/7.0) completeness (%) ^(a) 99.2 (98.6) 93.6 (60.2)number of unique 69,525 71,681 reflections redundancy ^(a) 4.0 (3.9) 6.8(5.0) monomers/asu 1 1 number of non- hydrogen atoms: protein 3069 3069ligand (type) ^(d) 23 (AMP) 13 (FLC) metal ions (zinc) 2 2glycosylations 56 56 waters 464 499 Wilson B 17.5 18.1 average B for:overall 26.4 25.7 macromolecule 24.3 23.5 ligand 34.0 29.8 solvent 40.339.6 Rwork/Rfree (%) ^(e, f) 14.0/18.4 15.4/20.1 rmsd bonds (Å) 0.0090.009 rmsd angles (°) 1.27 1.20 Ramachandran plot 99.7/0.3  99.5/0.5 (%): preferred or allowed/outliers residue range 24-402  24-402  PDB IDcode 4LQY 4LR2 ^(a) Values for the highest resolution bin are shown inparenthesis. ^(b) R_(sym) = Σ_(hkl) Σ_(i) |I_(i)(hkl) −<I(hkl)>|/Σ_(hkl) Σ_(i) I_(i)(hkl) ^(c) I/σI is the average intensity ofreflections in thin resolution bins divided by the average standarddeviation (sigma) of the same group of reflections. ^(d) AMP is5′-adenosine monophosphate. FLC is a citrate anion from thecrystallization conditions. ^(e) R_(work) = Σ||F_((obs))| −|F_((calc))||/Σ|F_((obs))| ^(f) R_(free) = as for Rwork, but calculatedfor 5.0% of the total reflections that were chosen at random and omittedfrom refinement.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of ameliorating vascular calcificationin a human subject having reduced ecto-nucleotidepyrophosphate/phosphodiesterase-1 (ENPP1) activity, said methodcomprising administering to said subject a therapeutically effectiveamount of a soluble polypeptide comprising an ENPP1 polypeptidecatalytic domain, wherein said soluble polypeptide lacks a domaincomprising 4 to 20 sequential aspartic acid residues, therebyameliorating said vascular calcification.
 2. A method of amelioratingvascular calcification in a human subject having a loss of functionmutation in the gene encoding ecto-nucleotidepyrophosphate/phosphodiesterase-1 (ENPP1), said method comprisingadministering to said subject a therapeutically effective amount of asoluble polypeptide comprising an ENPP1 polypeptide catalytic domain,wherein said soluble polypeptide lacks a domain comprising 4 to 20sequential aspartic acid residues, thereby ameliorating said vascularcalcification.
 3. The method of claim 1, wherein said solublepolypeptide comprises amino acid residues 96-925 of human ENPP1 [SEQ IDNO: 1].
 4. The method of claim 1, wherein said soluble polypeptide is afusion protein comprising an IgG Fc domain.
 5. The method of claim 3,wherein said soluble polypeptide is a fusion protein comprising an IgGFc domain.
 6. The method of claim 1, wherein said soluble polypeptidecomprises amino acid residues 93-925 of human ENPP1 [SEQ ID NO: 1]. 7.The method of claim 6, wherein said soluble polypeptide is a fusionprotein comprising an IgG Fc domain.
 8. The method of claim 2, whereinsaid soluble polypeptide comprises amino acid residues 96-925 of humanENPP1 [SEQ ID NO: 1].
 9. The method of claim 2, wherein said solublepolypeptide is a fusion protein comprising an IgG Fc domain.
 10. Themethod of claim 8, wherein said soluble polypeptide is a fusion proteincomprising an IgG Fc domain.
 11. The method of claim 2, wherein saidsoluble polypeptide comprises amino acid residues 93-925 of human ENPP1[SEQ ID NO: 1].
 12. The method of claim 11, wherein said solublepolypeptide is a fusion protein comprising an IgG Fc domain.
 13. Themethod of claim 1, wherein said polypeptide is administered parenterallyto said subject.
 14. The method of claim 2, wherein said polypeptide isadministered parenterally to said subject.
 15. The method of claim 1,wherein prior to administration of said soluble ENPP1 polypeptide, serumENPP1 activity is detected in said subject.
 16. The method of claim 2,wherein prior to administration of said soluble ENPP1 polypeptide, serumENPP1 activity is detected in said subject.
 17. The method of claim 1,wherein prior to administration of said soluble ENPP1 polypeptide, thelevel of at least one of serum Pi and serum PPi is detected in saidsubject.
 18. The method of claim 2, wherein prior to administration ofsaid soluble ENPP1 polypeptide, the level of at least one of serum Piand serum PPi is detected in said subject.
 19. The method of claim 1,wherein prior to administration of said soluble ENPP1 polypeptide, agenetic defect associated with ENPP1 dysfunction is detected in saidsubject.
 20. The method of claim 2, wherein prior to administration ofsaid soluble ENPP1 polypeptide, a genetic defect associated with ENPP1dysfunction is detected in said subject.